Production and use of derivatized homoserine lactones

ABSTRACT

The present invention provides analogues of autoinducer molecules that are derivatized to allow their attachment to other molecules and surfaces. Libraries of the autoinducer analogues are also contemplated. Also provided are methods for using the compounds of the invention to produce compositions, such as immunoconjugates, antibodies and vaccines, which are useful for treating and preventing disease states in a subject. The compositions of the invention are also useful in various assays, including assessing the autoinducer load in a subject.

BACKGROUND OF THE INVENTION

[0001] Autoinducers (“AIs”) are extracellular signal compounds used by avariety of bacteria to regulate cellular functions in response tochanges in population density. For example, light production by themarine symbiotic bacterium Vibrio fischeri is controlled in a populationdensity-responsive manner by the self-produced, membrane-permeableautoinducer, N-3-oxohexanoyl-L-homoserine lactone(N-3-oxohexanoyl-L-HSL; AI-1). AI-1 accumulates in a populationdensity-dependent manner during bacterial growth. When it reaches athreshold concentration, AI-1, via the autoinducer receptor andtranscriptional activator, LuxR, activates transcription of the luxoperon, luxICDABEG, which encodes autoinducer synthase (luxI) andluminescence enzymes. (Eberhard et al., Biochemistry 20: 2444-2449(1981); Engebrecht et al. Cell, 32: 773-781 (1983); Engebrecht et al.,Proc. Natl. Acad. Sci. USA 81: 4154-4158 (1984); Hanzelka et al., J.Bacteriol. 177: 815-817 (1995); Shadel et al, J. Bacteriol. 172:3980-3987 (1990); Slock et al., J. Bacteriol., 172:3974-3979 (1990);Swartzman et al., J. Bacteriol. 172: 6797-6802 (1990)). Theautoinduction mechanism in V. fischeri also involves, among otherregulatory aspects, an AI-1 mediated luxR negative autoregulation(Dunlap et al., J. Bacteriol. 170: 4040-4046 (1988); Dunlap et al., J.Bacteriol. 171: 3546-3552 (1989); Engebrecht et al., Genet. Eng. 8:31-44 (1986); Shadel et al., J. Bacteriol. 173: 568-574 (1991); Shadelet al., J. Biol. Chem. 267: 7690-7695 (1992)).

[0002] Long thought to be a regulatory mechanism unique to theluminescence system of V. fischeri and certain closely related marineluminous bacteria, autoinduction of gene expression recently has beenidentified in a wide variety of other bacteria (Fuqua et al., J.Bacteriol. 176:269-275 (1994)). The diversity of species usingautoinduction and the chemical and genetic similarities of theirautoinduction systems indicate that autoinduction is an evolutionaryconserved regulatory mechanism commonly used by bacteria to sense andrespond to population density.

[0003] All bacteria presently known to utilize AIs associate with higherorganisms, i.e., plants and animals, at some point during theirlifecycles. For example, Pseudomonas aeruginosa is an opportunisticpathogen in humans with cystic fibrosis. P. aeruginosa regulates variousvirulence determinants with AI (Davies et al, Science 280: 295 (1998)).Other examples of AI producing bacteria include Erwinia carotovora,Pseudomonas aureofaciens, Yersinia enterocolitica, Vibrio harveyi, andAgrobacterium tumefaciens. E. carotovora infects certain plants andcreates enzymes that degrade the plant's cell walls, resulting in whatis called “soft rot disease.” E. carotovora produces the autoinducerN-3-oxohexanoyl-L-HSL. Yersinia enterocolitica is a bacterium, whichcauses gastrointestinal disease in humans and has been reported toproduce an autoinducer. P. aureofaciens associates with the roots ofplants and produces antibiotics that block fungus growth in the roots.That antibiotic synthesis is under autoinducer control.

[0004] In addition to the known naturally occurring autoinducers, recentwork has focused on the synthesis and testing of synthetic analogues ofcertain autoinducers. For example, Bycroft et al., U.S. Pat. No.5,593,827 have synthesized a series of N-(β-ketocaproyl)-L-homoserinelactone derivatives. The homoserine lactone derivatives are activeautoinducers and control gene expression in certain organisms.Additionally, the autoinducer analogue N-(3-oxo-dodecanoyl)-homoserinelactone has been shown to inhibit the activity of P. aeruginosa (Pearsonet al., U.S. Pat. No. 5,591,872). Furthermore, autoinducer analoguesbased on a furanone ring structure have been shown to inhibit homoserinelactone regulated processes in microorganisms (Kjellberg et al., WO96/29392). Cao and coworkers have synthesized a series of N-acylhomoserine lactones and assessed their binding parameters andstructure-function relationship in the V. harveyi lux system. None ofthese references describes the synthesis of autoinducer analogues thatare suitable for attachment to other molecules and surfaces.

[0005] Autoinducer molecules are thought to have particular relevance inthe progression of cystic fibrosis (CF). CF is the most commoninheritable lethal disease among Caucasians. There are approximately25,000 CF patients in the U.S.A. The frequency of CF in several othercountries (e.g., Canada, United Kingdom, Denmark) is high (ranging from1 in 400 to 1 in 1,600 live births).

[0006] Chronic respiratory infections caused by mucoid Pseudornonasaeruginosa are the leading cause of high morbidity and mortality in CF.The initially colonizing P. aeruginosa strains are nonmucoid but in theCF lung they inevitably convert into the mucoid form. The mucoid coatingcomposed of the exopolysaccharide alginate leads to the inability ofpatients to clear the infection, even under aggressive antibiotictherapies. The emergence of the mucoid form of P. aeruginosa isassociated with further disease deterioration and poor prognosis.

[0007] The microcolony mode of growth of P. aeruginosa, embedded inexopolysaccharide biofilms in the lungs of CF patients (Lam et al.,Infect. Immun. 28: 546 (1980)), among other functions, plays a role inhindering effective opsonization and phagocytosis of P. aeruginosa cells(Pier et al., N. Engl. J. Med. 317: 793-8 (1987); Pier et al., Infect.Immun. 60: 4768-76 (1992)). Although CF patients can produce opsonicantibodies against P. aeruginosa antigens, in most cases phagocyticcells cannot effectively interact with such opsonins (Pressler et al.,Clin. Exp. Immunol. 90: 209-14 (1992); Pier et al., Science 249: 537-40(1990)). Physical hindrance caused by the exopolysaccharide alginate anda functionally important receptor-opsonin mismatch caused by chronicinflammation and proteolysis are contributing factors to the ineffectiveinteractions (Tosi et al., J. Infect. Dis. 162: 156-62 (1990)).Moreover, the biofilm prevents the effective delivery of exogenousantimicrobial agents to the microorganisms of the colony (de Beer etal., Appl. Environ. Microbiol. 60: 4339 (1994)).

[0008] Compounds and compositions facilitating the study ofautoinduction mechanisms, particularly biofilm formation, and which areeffective in disrupting biofilms or retarding their formation wouldrepresent a significant advance in the treatment of disease statesassociated with biofilms, such as CF. Quite surprisingly, the presentinvention provides such compounds and compositions.

SUMMARY OF THE INVENTION

[0009] Thus, in a first aspect, the present invention provides acompound having a structure according to Formula I:

[0010] wherein, R¹ is preferably a member selected from —H, —OH, and(═O); R² is preferably a member selected from reactive functionalgroups, alkyl groups terminally substituted with a reactive functionalgroup and internally substituted alkyl groups terminally substitutedwith a reactive functional group; X is preferably a member selected from—O—, —S— and —NH—; and X¹ and X² are preferably members independentlyselected from O and S.

[0011] In a second aspect, the present invention provides a compoundhaving the structure according to Formula II:

[0012] wherein, R¹ is preferably a member selected from H, OH, and (═O);and R² is preferably a member selected from reactive functional groups,alkyl groups terminally substituted with a reactive functional group andinternally substituted alkyl groups terminally substituted with areactive functional group.

[0013] In a third aspect, the present invention provides a compoundhaving a structure that is a member selected from:

[0014] wherein, m is preferably a number selected from 1 to 20,inclusive; n is preferably a number from 0 to 20, inclusive; and Z is areactive functional group.

[0015] In a fourth aspect, the invention provides an immobilizedcompound comprising a solid support to which is attached a moleculecomprising a structure according to Formula VI:

[0016] wherein, R¹ is preferably a member selected from —H, —OH, and(═O); R⁹ is preferably a member selected from alkyl groups andsubstituted alkyl groups and is attached to a solid support; X ispreferably a member selected from —O—, —S— and —NH—; and X¹ and X² arepreferably members independently selected from O and S.

[0017] In a fifth aspect the present invention provides an immunogenicconjugate comprising a target component including a structure accordingto Formula VI, above, wherein R⁹ is attached to a carrier molecule.

[0018] In a sixth aspect, the invention provides a library of compoundshaving a structure according to Formula I, wherein the library comprisesa first compound according to Formula I and a second compound accordingto Formula I, wherein the first compound differs from the secondcompound in the identity of a member selected from R¹, R⁹, X, X¹, X² andcombinations thereof.

[0019] In a seventh aspect, the invention provides a kit for detectingan autoinducer in a sample. The kit includes, an antibody that bindsspecifically to the autoinducer and directions for using the antibody todetect the autoinducer.

[0020] In a eighth aspect, the invention provides a method of detectingan autoinducer in a sample. The method includes, the steps of: (a)contacting the sample with an antibody that specifically binds to theautoinducer; and (b) determining whether the sample contains theautoinducer.

[0021] In an ninth aspect, the present invention provides a method ofmonitoring the amount of autoinducer in a patient treated with an agentthat inhibits the growth of an organism producing the autoinducer. Themethod includes: (a) providing a sample from the patient treated withthe growth inhibiting agent; (b) contacting the sample with an antibodythat specifically binds to an autoinducer; (c) forming a complex betweenthe antibody and the autoinducer; and (d) determining the amount ofautoinducer in the patient sample by detecting the antibody or theantibody-autoinducer complex and comparing the amount of antibodydetected in the patient sample to a standard curve, thereby monitoringthe amount of autoinducer in the patient.

[0022] In a tenth aspect, the invention provides a method of isolatingan autoinducer. The method includes the steps of: (a) providing a samplecomprising the autoinducer; (b) contacting the sample with an antibodythat specifically binds to the autoinducer, thereby forming anantibody-autoinducer complex; and (c) isolating the autoinducer byisolating the antibody-autoinducer complex.

[0023] In a eleventh aspect, the invention provides a method ofdetecting an antibody that specifically binds to an autoinducer. Themethod include the steps of: (a) providing a sample; (b) contacting thesample with a peptide that specifically binds to the antibody; and (c)detecting the antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1 is an exemplary synthetic scheme for the preparation ofcompound 4.

[0025]FIG. 2 is an exemplary synthetic scheme for the preparation ofcompound 13.

[0026]FIG. 3 is a graphical display of the ELISA results vs. dilutionfor antibodies of the invention.

DETAILED DESCRIPTION OF THE INVENTION AND THE PREFERRED EMBODIMENTS

[0027] Introduction

[0028] The present invention provides, for the first time, an array ofautoinducer analogues that are derivatized with a reactive group thatallows the conjugation of the analogues to surfaces and to othermolecules. The reactive autoinducer analogues are useful for thepreparation of materials such as affinity chromatography supports,immunogenic conjugates, autoinducer libraries, and the like. Theimmobilized or conjugated autoinducers are used for, among other things,raising mono- and poly-clonal antibodies against autoinducers, isolatingcellular receptors that interact with autoinducers and detectingmolecules (e.g., antibodies) that interact with autoinducers. Theefficacy of art-recognized anti-infective agents can be increased byconjugating them to the reactive autoinducer analogues described herein.The reactive autoinducer analogues are easily synthesized, often fromcommercially available precursors. Moreover, as the fields ofbioconjugate chemistry, peptide synthesis and oligonucleotide synthesisprovide a wealth of techniques for coupling small reactive molecules tosurfaces, polymers, (e.g., biomolecules), and to other small molecules,the ready availability of autoinducers derivatized to allow theirconjugation to other species provides access to a wealth of compoundsand compositions of diagnostic and therapeutic use, as well as compoundsuseful in elucidating the structure and mechanism of action ofautoinducers.

[0029] Definitions

[0030] The terms used to describe the preferred embodiments of thepresent invention will generally have their art-recognized meanings. Thedefinitions offered below are intended to supplement, not supplant, theart-recognized meanings.

[0031] “RAA,” as used herein refers to “reactive autoinducer analogue.”

[0032] The term “independently selected” is used herein to indicate thatthe groups so described can be identical or different

[0033] “Autoinducer,” as used herein includes molecules that arepreferably a component of a system that regulates intercellular activityin response to environmental conditions and includes extracellularsignal molecules. Exemplary autoinducers include, for example, theacylated homoserine lactones of microorganisms, such as Vibrio harveyiand P. aeruginosa and an array of structurally analogous compounds. Theterm “autoinducer” is a generic term that also incorporates, for manypurposes, the term “autoinducer analogue” and includes the reactiveautoinducer analogues of the invention.

[0034] “Autoinducer analogue,” is generally used herein to refer to aspecies of the invention. The compounds of the invention are preferablystructurally analogous to known autoinducer molecules, with theexception that the molecules of the invention include within theirstructure a reactive functional group that allows them to be tethered toother molecules and/or surfaces. “Autoinducer analogues” alsoencompasses, the compounds of the invention that have been tethered toanother molecule or surface via reaction of their reactive functionalgroup.

[0035] The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native or crude state. A materialthat is the predominant species present in a preparation issubstantially purified. Purity and homogeneity are typically determinedusing analytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography (HPLC). Whenused in combination with proteins and nucleic acids, the term “purified”denotes that a nucleic acid or protein gives rise to essentially oneband in an electrophoretic gel. Particularly, it means that the nucleicacid or protein is at least 85% pure, more preferably at least 95% pure,and most preferably at least 99% pure.

[0036] “Nucleic acid” refers to deoxyribonucleotides or ribonucleotidesand polymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

[0037] Unless otherwise indicated, a particular nucleic acid sequencealso implicitly encompasses conservatively modified variants thereof(e.g., degenerate codon substitutions) and complementary sequences, aswell as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

[0038] The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer. These termsalso encompass the term “antibody.”

[0039] The term “amino acid” refers to naturally occurring and syntheticamino acids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine stilfoxide, methionine methylsulfonium. Such analogs-have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic-chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

[0040] The term “immunoassay” refers to an assay that uses an antibodyto specifically bind an antigen. The immunoassay is characterized by,for example, the use of specific binding properties of a particularantibody to isolate, target, and/or quantify the antigen.

[0041] The phrase “specifically (or selectively) binds” to an antibodyor “specifically (or selectively) immunoreactive with,” when referringto an autoinducer or other substance (e.g., protein, peptide),delineates a binding reaction that is determinative of the presence ofthe autoinducer in a heterogeneous population of the autoinducer andother substances, preferably biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to a particularautoinducer or autoinducer analogue. The antibody preferably does notsubstantially bind in a significant amount to other substances presentin the sample. Specific binding to an antibody under such conditions mayrequire an antibody that is selected for its specificity for aparticular autoinducer. For example, polyclonal antibodies raised to anautoinducer can be selected to obtain only those polyclonal antibodiesthat are specifically immunoreactive with the autoinducer and not withother autoinducers or other substances. This selection can be achievedby subtracting out antibodies that cross-react with molecules such asother autoinducers.

[0042] By “host cell” is meant a cell that contains an expression vectorand supports the replication or expression of the expression vector.Host cells may be prokaryotic cells such as E. coli, or eukaryotic cellssuch as yeast, insect, amphibian, or mammalian-cells such as CHO, HeLaand the like, e.g., cultured cells, explants, and cells in vivo. A “hostcell” also refers to a cell, such as a hybridoma, that-produces anantibody.

[0043] “Biological sample,”, as used herein, includes a sample ofbiological tissue or fluid that contains an autoinducer or ananti-autoinducer antibody. Such samples include, but are not limited to,tissue isolated from humans. Fluids include blood, serum, plasma, urine,sputum, cerebral spinal fluid, and other such fluids. Biological samplesmay also include sections of tissues such as frozen sections taken forhistologic purposes. A biological sample is typically obtained from aeukaryotic organism, preferably eukaryotes such as fungi, plants,insects, protozoa, birds, fish, reptiles, and preferably a mammal suchas rat, mice, cow, dog, guinea pig, or rabbit, and most preferably aprimate such as chimpanzees or humans.

[0044] The term “alkyl” includes branched or unbranched, saturated orunsaturated, monovalent hydrocarbon radical, generally having from about1-30 carbons and preferably, from 4-20 carbons and more preferably from6-18 carbons. When the alkyl group has from 1-6 carbon atoms, it isreferred to as a “lower alkyl.” Suitable alkyl radicals include, forexample, structures containing one or more methylene, methine and/ormethyne groups. Branched structures have a branching motif similar toi-propyl, t-butyl, i-butyl, 2-ethylpropyl, etc. As used herein, the termencompasses “substituted alkyl.”

[0045] “Substituted alkyl” inlcudes alkyl as just described includingone or more substituents such as lower alkyl, aryl, acyl, halogen (i.e.,alkylhalos, e.g., CF₃), hydroxy, amino, alkoxy, alkylamino, acylamino,thioamido, acyloxy, aryloxy, aryloxyalkyl, mercapto, thia, aza, oxo,both saturated and unsaturated cyclic hydrocarbons, heterocycles and thelike. These groups may be attached to any carbon or substituent of thealkyl moiety. Additionally, these groups may be pendent from, orintegral to, the alkyl chain.

[0046] The term “aryl” is used herein includes an aromatic substituentwhich may be a single aromatic ring or multiple aromatic rings which arefused together, linked covalently, or linked to a common group such as amethylene or ethylene moiety. The common linking group may also be acarbonyl as in benzophenone. The aromatic ring(s) may include phenyl,naphthyl, biphenyl, diphenylmethyl and benzophenone among others. Theterm “aryl” encompasses “substituted aryl.”

[0047] “Substituted aryl” includes aryl as just described including oneor more functional groups such as lower alkyl, acyl, halogen, alkylhalos(e.g. CF₃), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy,phenoxy, mercapto and both saturated and unsaturated cyclic hydrocarbonswhich are fused to the aromatic ring(s), linked covalently or linked toa common group such as a methylene or ethylene moiety. The linking groupmay also be a carbonyl such as in cyclohexyl phenyl ketone. The term“substituted aryl” encompasses “substituted arylalkyl.”

[0048] The term “arylalkyl” is used herein to refer to a subset of“aryl” in which the aryl group is attached to another group by an alkylgroup as defined herein.

[0049] “Substituted arylalkyl” defines a subset of “substituted aryl”wherein the substituted aryl group is attached to another group by analkyl group as defined herein.

[0050] The term “acyl” is used to describe a ketone substituent, —C(O)R,where R is alkyl or substituted alkyl, aryl or substituted aryl asdefined herein.

[0051] The term “halogen” is used herein to refer to fluorine, bromine,chlorine and iodine atoms.

[0052] The term “hydroxy” is used herein to refer to the group —OH.

[0053] The term “amino” is used to describe primary amines, —NRR′,wherein R and R′ are independently H, alkyl, aryl or substitutedanalogues thereof. “Amino” encompasses “alkylamino” denoting secondaryand tertiary amines and “acylamino” describing the group RC(O)NR′.

[0054] The term “alkoxy” is used herein to refer to the —OR group, whereR is alkyl, aryl, or substituted analogues thereof. Suitable alkoxyradicals include, for example, methoxy, ethoxy, t-butoxy, etc.

[0055] The term “acyloxy” is used interchangeably with “ester” todescribe an organic radical derived from an organic acid by the removalof the acidic hydrogen. Simple acyloxy groups include, for example,acetoxy, and higher homologues derived from carboxylic acids such asethanoic, propanoic, butanoic, etc. The acyloxy moiety may be orientedas either a forward or reverse ester (i.e. RC(O)OR′ or R′OC(O)R),wherein one or both of R and R′ is a component of the parent moleculehaving the “acyloxy” group as a substituent.

[0056] As used herein, the term “aryloxy” denotes aromatic groups, whichare linked to another group directly through an oxygen atom. Exemplaryaryloxy groups include phenoxy, substituted phenoxy, benzyloxy,phenethyloxy. “Aryloxy” encompasses “substituted aryloxy” moieties inwhich the aromatic group is substituted as described above for“substituted aryl.”

[0057] As used herein “aryloxyalkyl” defines aromatic groups attached,through an oxygen atom to an alkyl group, as defined herein. The term“arylox yalkyl” encompasses “substituted aryloxyalkyl” moieties in whichthe aromatic group is substituted as described for “substituted aryl.”

[0058] As used herein, the term “mercapto” defines moieties of thegeneral structure R—S—R′ wherein R and R′ are the same or different andone or more or R and R′ is a component of the parent molecule having themercapto group as a substituent.

[0059] The term “saturated cyclic hydrocarbon” denotes carbocyclicgroups such as, for example, cyclopropyl, cyclobutyl, cyclopentyl, etc.,and substituted analogues of these structures. These cyclic hydrocarbonscan be single- or multi-ring structures.

[0060] The term “unsaturated cyclic hydrocarbon” is used to describe anon-aromatic carbocycle with at least one double bond, such as, forexample, cyclopentene, cyclohexene, etc. and substituted analoguesthereof. These cyclic hydrocarbons can be single- or multi-ringstructures.

[0061] The term “heteroaryl” as used herein refers to aromatic rings inwhich one or more carbon atoms of the aromatic ring(s) are replaced by aheteroatom such as nitrogen, oxygen or sulfur. Heteroaryl refers tostructures, which may be a single aromatic ring, multiple aromaticring(s), or one or more aromatic rings coupled to one or morenon-aromatic ring(s). In structures having multiple rings, the rings canbe fused together, linked covalently, or linked to a common group suchas a methylene or ethylene moiety. The common linking group may also bea carbonyl as in phenyl pyridyl ketone. As used herein, rings such asthiophene, pyridine, isoxazole, phthalimide, pyrazole, indole, furan,etc. or benzo-fused analogues of these rings are defined by the term“heteroaryl.”

[0062] “Heteroarylalkyl” defines a subset of “heteroaryl” wherein analkyl group, as defined herein, links the heteroaryl group to the parentmolecule.

[0063] “Substituted heteroaryl” refers to heteroaryl as just describedwherein the heteroaryl nucleus is substituted with one or morefunctional groups such as lower alkyl, acyl, halogen, alkylhalos (e.g.CF₃), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, mercapto,etc. Thus, substituted analogues of heteroaromatic rings such asthiophene, pyridine, isoxazole, phthalimide, pyrazole, indole, furan,etc. or benzo-fused analogues of these rings are defined by the term“substituted heteroaryl.”

[0064] “Substituted heteroarylalkyl” refers to a subset of “substitutedheteroaryl” as described above in which an alkyl group, as definedherein, links the heteroaryl group to the parent molecule.

[0065] The term “heterocyclic” is used herein to describe a monovalentsaturated or unsaturated non-aromatic group having a single ring ormultiple condensed rings from 1-12 carbon atoms and from 1-4 heteroatomsselected from nitrogen, sulfur or oxygen within the ring. Suchheterocycles are, for example, tetrahydrofuran, morpholine, piperidine,pyrrolidine, etc.

[0066] The term “substituted heterocyclic” as used herein describes asubset of “heterocyclic” wherein the heterocycle nucleus is substitutedwith one or more functional groups such as lower alkyl, acyl, halogen,alkylhalos (e.g. CF₃), hydroxy, amino, alkoxy, alkylamino, acylamino,acyloxy, mercapto, etc.

[0067] The term “heterocyclicalkyl” defines a subset of “heterocyclic”wherein an alkyl group, as defined herein, links the heterocyclic groupto another group.

[0068] The Compounds

[0069] In a first aspect, the present invention provides a moleculehaving a structure according to Formula I:

[0070] R¹ is preferably a member selected from —H, —OH, and (═O); R² ispreferably a member selected from H, reactive functional groups, alkylgroups terminally substituted with a reactive functional group andinternally substituted alkyl groups terminally substituted with areactive functional group; X is preferably a member selected from —O—,—S— and —NH—; and X¹ and X² are preferably members independentlyselected from O and S. In another embodiment, X¹ is preferably an aminederivative such as an alkyl-, or hydroxyl-amine. In yet anotherembodiment, X is preferably an alkyl-, acyl-, or hydroxyl-amine.

[0071] In a second aspect, the present invention provides compoundshaving a structure according to Formula II:

[0072] R¹ is preferably a member selected from H, OH, and (═O); and R²is preferably a member selected from reactive functional groups, alkylgroups terminally substituted with a reactive functional group andinternally substituted alkyl groups terminally substituted with areactive functional group.

[0073] The following discussion is generally applicable to each of theaspects of the invention discussed above. In a preferred embodiment, R²is an internally substituted alkyl group terminally substituted with areactive functional group. The alkyl group can be straight- orbranched-chain and it can include regions of unsaturation. Theinternally substituted alkyl group can be functionalized at withsubstituents that interrupt the alkyl chain, with substituents pendentfrom the alkyl chain or a combination thereof. Substantially any type ofsubstituent and/or substitution pattern, which can be incorporated intoan alkyl group is useful in practicing the present invention.Furthermore, the alkyl chain described herein is not limited in thenumber or identity of the substitutions that it bears. Those of skill inthe art are able to select appropriate substitutions for a particularapplication. Useful internal substitutions include, for example, —O—,—NR—, —S—, double bonds (e.g., C═C, C═N, C═O) and the like. Preferredsubstituents pendent from the alkyl chain include, for example, —OH—,—NRR′, —NRR′R″^(⊕), —SH, —COOR and the like. As used herein, R, R′ andR″ represent H, unsubstituted and substituted alkyl and aryl groups.Presently preferred internal substituents include —OH, (═O) andcombinations thereof.

[0074] In preferred embodiments of each of the above-described aspectsof the present invention, the alkyl and the internally substituted alkylgroups are members selected from C₁-C₂₀ saturated straight-chain, C₁-C₂₀saturated branched-chain, C₁-C₂₀ unsaturated straight-chain, C₁-C₂₀unsaturated branched-chain alkyl and internally substituted alkylgroups, and more preferably, the C₅-C₁₀ analogues of each of theabove-recited structures.

[0075] In still further preferred embodiments, R² has the structure setforth in Formula III:

(CH₂)_(n)—R⁷  (III)

[0076] wherein, R⁷ is a reactive functional group; and n is a numberfrom 1 to 20, inclusive, and more preferably, a number from 2 to 9,inclusive.

[0077] In yet another preferred embodiment, R² has a structure accordingto Formula IV:

[0078] wherein, R⁷ is a reactive functional group; and q and s arenumbers independently selected from 1 to 20, inclusive, more preferably,from 2 to 9, inclusive.

[0079] In a third aspect, the present invention provides compoundshaving a structure according to the structures displayed in Table 1.TABLE 1

[0080] In each of the compounds displayed in Table 1, Z represents areactive functional group and m and n are preferably numbersindependently selected from 1 to 20, inclusive, more preferably from 2to 9, inclusive.

[0081] The compounds of the invention can be prepared as a single isomer(e.g., enantiomer, cis-trans, positional, diastereomer) or as a mixtureof isomers. In a preferred embodiment, the compounds are prepared assubstantially a single isomer. Methods of preparing substantiallyisomerically pure compounds are known in the art. For example,enantiomerically enriched mixtures and pure enantiomeric compounds areprepared by using synthetic intermediates that are enantiomerically purein combination with reactions that either leave the stereochemistry at achiral center unchanged or result in its complete inversion.Alternatively, the final product or intermediates along the syntheticroute can be resolved into a single stereoisomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose andappropriate method for a particular situation. See, generally, Furnisset al. (eds.), VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY5^(TH) ED., Longman Scientific and Technical Ltd., Essex, 1991, pp.809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

[0082] Reactive Functional Groups

[0083] The compounds of the invention bear a reactive functional group,which can be located at any position on the alkyl chain, but which isgenerally located at a terminal position of the molecule. Reactivegroups and classes of reactions useful in practicing the presentinvention are generally those that are well known in the art ofbioconjugate chemistry. Currently favored classes of reactions availablewith reactive autoinducer analogues are those which proceed underrelatively mild conditions. These include, but are not limited tonucleophilic substitutions (e.g., reactions of amines and alcohols withacyl halides, active esters), electrophilic substitutions (e.g., enaminereactions) and additions to carbon-carbon and carbon-heteroatom multiplebonds (e.g., Michael reaction, Diels-Alder addition). These and otheruseful reactions are discussed in, for example, March, ADVANCED ORGANICCHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson,BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney etal., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198,American Chemical Society, Washington, D.C., 1982.

[0084] Useful reactive functional groups include, for example:

[0085] (a) carboxyl groups and various derivatives thereof including,but not limited to, N-hydroxysuccinimide esters, N-hydroxybenztriazoleesters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters,alkyl, alkenyl, alkynyl and aromatic esters;

[0086] (b) hydroxyl groups which can be converted to esters, ethers,aldehydes, etc.

[0087] (c) haloalkyl groups wherein the halide can be later displacedwith a nucleophilic group such as, for example, an amine, a carboxylateanion, thiol anion, carbanion, or an alkoxide ion, thereby resulting inthe covalent attachment of a new group at the site of the halogen atom;

[0088] (d) dienophile groups which are capable of participating inDiels-Alder reactions such as, for example, maleimido groups;

[0089] (e) aldehyde or ketone groups such that subsequent derivatizationis possible via formation of carbonyl derivatives such as, for example,imines, hydrazones, semicarbazones or oximes, or via such mechanisms asGrignard addition or alkyllithium addition;

[0090] (f) sulfonyl halide groups for subsequent reaction with amines,for example, to form sulfonamides;

[0091] (g) thiol groups, which can be converted to disulfides or reactedwith acyl halides;

[0092] (h) amine or sulfhydryl groups, which can be, for example,acylated, alkylated or oxidized;

[0093] (i) alkenes, which can undergo, for example, cycloadditions,acylation, Michael addition, etc; and

[0094] (j) epoxides, which can react with, for example, amines andhydroxyl compounds.

[0095] The reactive functional groups can be chosen such that they donot participate in, or interfere with, the reactions necessary toassemble the reactive autoinducer analogue. Alternatively, a reactivefunctional group can be protected from participating in the reaction bythe presence of a protecting group. Those of skill in the art willunderstand how to protect a particular functional group from interferingwith a chosen set of reaction conditions. For examples of usefulprotecting groups, See Greene et al., PROTECTIVE GROUPS IN ORGANICSYNTHESIS, John Wiley & Sons, New York, 1991.

[0096] In a preferred embodiment, the reactive functional group isselected from the groups —OR³, —NHR⁴, —COR⁵, —SH and —CH₂X³ In thesegroups, —OR³ is selected from hydroxyl, alkyl sulfonate or arylsulfonate groups. R⁴ is selected from H, C₁-C₆ alkyl, C₁-C₆ substitutedalkyl, aryl and substituted aryl groups. R⁵ is selected from H, X³ and—OR⁶. R⁶ is a species selected such that —OR⁶ is a leaving group. X³ isa halogen.

[0097] In a further preferred embodiment R⁶ is selected from alkyl,substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclyl and substituted heterocyclyl groups.

[0098] In a still further preferred embodiment, R³ has a structureaccording to Formula V:

[0099] wherein, R⁸ is a member selected from alkyl, substituted alkyl,aryl and substituted aryl groups.

[0100] In yet another preferred embodiment, the reactive functionalgroup is selected from —COOH, —OH, —NH₂, and —SH.

[0101] Synthesis

[0102] The compounds of the invention are synthesized by an appropriatecombination of generally well known synthetic methods. Methods ofsynthesizing the compounds of the invention are both readily apparentand accessible to those of skill in the relevant art. The discussionbelow is offered to illustrate certain of the diverse methods availablefor use in assembling the compounds of the invention, it is not intendedto define the scope of reactions or reaction sequences that are usefulin preparing the compounds of the present invention.

[0103] One method of synthesizing compounds of the invention is setforth in Scheme 1 (FIG. 1). A precursor dicarboxylic acid is activatedat a single carboxylic acid by conversion of the dicarboxylic acid tothe corresponding cyclic anhydride 1. The cyclic anhydride issubsequently reacted with benzyl alcohol. The reaction of the cyclicanhydride with benzyl alcohol provides a dicarboxylic acid derivative 2in which only one of the carboxylic acid moieties is protected. Theunprotected carboxylic acid group is coupled to the exocyclic aminegroup of homoserine lactone using a dehydrating reagent in amodification of art-recognized peptide chemistry to produce lactonederivative 3. The benzyl alcohol moiety is removed by hydrogenolysisusing a Pd/C catalyst to afford acid 4.

[0104] Another method for synthesizing compounds of the invention is setforth in FIG. 2 (Scheme 2). An alkenyl carboxylic acid 4 is protected bybenzylation, forming 5. The alkene group of the protected acid ishydroborated and oxidized, affording alcohol 6. The alcohol is furtheroxidized to the corresponding carboxylic acid 7, which is subsequentlyactivated by conversion to an acyl chloride 8. The acyl chloride isreacted with a compound capable of providing a carbon atom chain of adesired length and which also includes a carboxylic acid or a precursorthereof 10. Decarboxylation of 10 affords a monoprotected dioic acid 11.The monoprotected acid is coupled to the exocyclic amine of homoserinelactone to provide compound 12, which is debenzylated, affording 13.

[0105] A number of variations on the basic schemes set forth above areuseful in synthesizing the compounds of the invention. For example, thesynthesis of certain compounds of the invention requires a dicarboxylicacid having a carbon backbone that is either too long or too short toproduce useful yields of a cyclized anhydride. Dicarboxylic acids thatcannot be cyclized, can be monoprotected. Numerous methods allowing themonoprotection of a dicarboxylic acid are known in the art. In oneexemplary method, a single carboxylic acid moiety is reacted at theexpense of the other acid (see, for example, Albert et al., Synthesis635 (1987)). In another method, both of the carboxylic acid groups areprotected as esters and a reagent that predominantly cleaves one esteris utilized to form the monoprotected adduct. Exemplary cleavingreagents include, but are not limited to, barium hydroxide (Inoue etal., Tetrahedron Lett. 4063 (1977)) and esterases (Ohno et al.,Tetrahedron 40:145 (1984)). Many other means of monoprotecting ahomodifunctional molecule are known in the art. For example, see,generally, Greene et al., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 2^(ND)ED., Wiley-Interscience, 1991.

[0106] Following the monoprotection of the dicarboxylic acid, theremaining carboxylic acid moiety is coupled to the exocyclic amine ofthe heterocyclic portion of the molecule. Numerous reagents andcarboxylic acid derivatives are useful in activating the carboxylic acidmoiety for reaction with the amine. Carboxylic acids can be activated byconversion to, for example, acyl halides, acid anhydrides, esters (e.g.,methyl) and active esters (e.g., N-hydroxysuccinimide). For example,see, generally, Sandler et al., ORGANIC FUNCTIONAL GROUP PREPARATIONS2^(ND) ED., Chapter 11, Academic Press, Inc., Dan Diego, 1983.

[0107] The cyclic portion of the molecule can be obtained fromcommercial sources (e.g., Aldrich, Sigma, Fluka). For example, bothhomocysteine thiolactone hydrochloride and homoserine lactonehydrobromide are commercially available (Aldrich). Alternatively,appropriate five-member heterocycles can be prepared by methods known inthe art (see, generally, March, ADVANCED ORGANIC CHEMISTRY 3^(RD) ED.,Wiley-Interscience, New York, 1985 and Katritzky, HANDBOOK OFHETEROCYCLIC CHEMISTRY, Pergamon Press Ltd., Oxford, 1985). Thepreparation of five-member heterocyclic ring systems is a well developedfield of endeavor. The techniques necessary to prepare an array offive-member heterocyclic systems useful in preparing the compounds ofthe present invention are known to and easily practiced by those ofskill in the art of organic synthesis.

[0108] An appropriately functionalized five-member cyclic lactam can beprepared by, for example, cyclizing an amino acid, such as2,4-diaminobutyric acid (see, e.g., Bladé-Font, Tetrahedron Lett. 21:2443 (1980). Many other methods, including cyclizing halo amides,hydrocarboxylation of unsaturated amines and reaction between cyclicketones an hydrazoic acidcan be used to prepare useful lactamderivatives.

[0109] The lactones, thiolactones and lactams of the invention can beconverted to derivatives in which the carbonyl oxygen is replaced with asulfur or amine group using methods known in the art. For example, thecarbonyl oxygen of lactones, thiolactones and lactams can be replaced bya sulfur atom using a reagent, such as bis(tricyclohexyltin)sulfide andBCl₃ (see, for example, Pederson et al., Bull. Soc. Chim. Belges 87: 229(1978); Pederson et al., Bull. Soc. Chim. Belges 87: 293 (1978); andGhattas et al., Sulfur Lett. 1: 69(1982)).

[0110] It will be apparent to those of skill in the art that usefulsynthetic schemes can diverge from those set forth in Schemes 1 and 2 ina number of ways. In one example, a heterobifunctional compound having asingle carboxylic acid moiety and a non-carboxylic acid moiety can beincorporated into the structure of the compounds of the invention. Forexample, a compound such as an co-amino acid can be protected at theamine position and utilized in an otherwise unchanged method accordingto Schemes 1 and 2. Following assembly of the desired molecule anddeprotection of the amine group, the product bears a reactive aminefunctionality, which allows for its attachment to another molecule orstructure. Similar schemes utilizing carboxylic acids with, for example,ω-hydroxyl, -thiol, -disulfide, -carbonyl, -diene, -dienophile, and thelike can be readily practiced. Furthermore, it will be apparent to thoseof skill in the art that the non-carboxylic acid group need not be atthe co and can be located at other positions on the chain.

[0111] Conjugated Autoinducer Analogs

[0112] In another preferred embodiment, the present invention providesan immobilized compound comprising a molecule, or a solid support towhich is attached a molecule comprising a structure according to FormulaVI:

[0113] In Formula VI, R¹ is preferably a member selected from —H, —OH,and (═O). R⁹ is preferably a member selected from alkyl groups andsubstituted alkyl groups. X is preferably a member selected from —O—,—S— and —NH—. X¹ and X² are preferably members independently selectedfrom O and S. In-another preferred embodiment, X¹ is an amine derivativesuch as an alkyl-, or -hydroxyl-amine. In yet another embodiment, X isan alkyl-, acyl-, or hydroxyl-amine.

[0114] In the interest of clarity, the discussion below focuses on theattachment of autoinducer molecules to solid supports. It will beapparent to those of skill in the art that this discussion alsoencompasses embodiments of the invention in which the autoinducer isattached to a species other than a solid support, such as a soluble oran insoluble molecule.

[0115] The compound of the invention can be immobilized on substantiallyany polymer, biomolecule, and solid or semi-solid material having anyuseful configuration. When the support is a solid or semi-solid,preferred solid supports include beads, particles, membranes,substantially planar surfaces and combinations thereof formulated frommaterials including silica, metal, plastic and combinations thereof.

[0116] According to the present invention, the surface of a solidsupport is functionalized with a compound of the invention (RAA) byreacting a RAA with a reactive group on the surface of the solidsupport, thereby derivatizing the solid support with one or moreautoinducer analogues. Reactive groups which can be used in practicingthe present invention are discussed in detail above and include, forexample, amines, hydroxyl groups, carboxylic acids, carboxylic acidderivatives, alkenes, sulfhydryls, siloxanes, etc.

[0117] A large number of solid supports appropriate for practicing thepresent invention are available commercially. Useful commerciallyavailable solid supports include, for example, peptide synthesis resins,both with and without attached amino acids and/or peptides (e.g.,alkoxybenzyl alcohol resin, aminomethyl resin, aminopqlystyrene resin,-benzhydrylamine resin, etc. (Bachem)), functionalized controlled poreglass (BioSearch Technologies), ion exchange media (Aldrich),functionalized membranes (e.g., —COOH membranes; Ashai Chemical Co.,Asahi Glass Co., and Tokuyama Soda Co.), and the like.

[0118] Moreover, for applications in which an appropriate solid supportis not commercially available, a wide variety of reaction types isavailable for the functionalization of a solid support surface. Forexample, supports constructed of a plastic such as polypropylene, can besurface derivatized by chromic acid oxidation, and subsequentlyconverted to hydroxylated or aminomethylated surfaces. Thefunctionalized support is then reacted with a RAA of complementaryreactivity such as a RAA active ester, acid chloride or sulfonate ester,for example. Supports made from highly crosslinked divinylbenzene can besurface derivatized by chloromethylation and subsequent functional groupmanipulation. Additionally, functionalized substrates can be made frometched, reduced polytetrafluoroethylene

[0119] When the support is constructed of a siliceous material such asglass, the surface can be derivatized by reacting the surface Si—OH,SiO—H, and/or Si—Si groups with a functionalizing reagent.

[0120] In a preferred embodiment, wherein the substrates are made fromglass, the covalent bonding of the reactive group to the glass surfaceis preferably achieved by conversion of groups on the substrate'ssurface by a silicon modifying reagent such as:

(R^(a)O)₃—Si—R^(b)—X^(a)  (VII)

[0121] where R^(a) is preferably an alkyl group, such as methyl orethyl, R^(b) is a linking group between silicon and X^(a), and X^(a) isa reactive group or a protected reactive group.

[0122] In another preferred embodiment, the reagent used tofunctionalize the solid support provides for more than one reactivegroup per each reagent molecule. Using reagents, such as VIII, below,each reactive site on the substrate surface is, in essence, “amplified”to two or more functional groups:

(R^(a)O)₃—Si—R^(b)—(X^(a))_(n)  (VIII).

[0123] where R^(a) is preferably an alkyl group (e.g., methyl, ethyl),R^(b) is a linking group between silicon and X^(a), X^(a) is a reactivegroup or a protected reactive group and n is preferably an integerbetween 2 and 50, and more preferably between 2 and 20. Silanederivatives having halogens or other leaving groups beside the displayedalkoxy groups of VII and VIII are also useful in the present invention.

[0124] The amplification of an autoinducer by its attachment to asilicon-containing substrate is intended to be exemplary of the generalconcept of autoinducer amplification. This amplification strategy isequally applicable to other aspects of the invention in which anautoinducer analogue is attached to another molecule or solid support.

[0125] A number of siloxane functionalizing reagents can be used, forexample:

[0126] 1. Hydroxyalkyl siloxanes (Silylate surface, functionalize withdiborane, and H₂O₂ to oxidize to the alcohol)

[0127] a. allyl trichlorosilane→→3-hydroxypropyl

[0128] b. 7-oct-1-enyl trichlorchlorosilane→→8-hydroxyoctyl

[0129] 2. Diol (dihydroxyalkyl) siloxanes (silylate surface andhydrolyze to diol)

[0130] a. (glycidyl trimethoxysilane→→(2,3-dihydroxypropyloxy)propyl

[0131] 3. Aminoalkyl siloxanes (amines requiring no intermediatefunctionalizing step)

[0132] a. 3-aminopropyl trimethoxysilane→aminopropyl

[0133] 4. Dimeric secondary aminoalkyl siloxanes

[0134] a. bis (3-trimethoxysilylpropyl) amine→bis(silyloxylpropyl)amine.

[0135] It will be apparent to those of skill in the art that an array ofsimilarly useful functionalizing chemistries are available when supportcomponents other than siloxanes are used. Thus, for example alkylthiols, functionalized as discussed above in the context ofsiloxane-modifying reagents, can be attached to metal films andsubsequently reacted with an RAA to produce the immobilized compound ofthe invention.

[0136] R groups of use for R^(b) in the above described embodiments ofthe present invention include, but are not limited to, alkyl,substituted alkyl, aryl, arylalkyl, substituted aryl, substitutedarylalkyl, acyl, halogen, hydroxy, amino, alkylamino, acylamino, alkoxy,acyloxy, aryloxy, aryloxyalkyl, mercapto, saturated cyclic hydrocarbon,unsaturated cyclic hydrocarbon, heteroaryl, heteroarylalkyl, substitutedheteroaryl, substituted heteroarylalkyl, heterocyclic, substitutedheterocyclic and heterocyclicalkyl groups and combinations thereof.

[0137] The immobilized constructs of the invention can also include aspacer moiety between the reactive group of the solid support and theautoinducer analogue. The properties of the linker between the solidsupport and the solid support reactive group and the linker between thesolid support reactive group and the autoinducer analogue havestructures and chemical compositions that are independently selectedfrom a large array of stable and cleavable structures. For the purposeof illustration, the discussion below focuses on the linker between thesolid support and the solid support reactive group. The discussion isalso generally relevant to a linker, if present, between the solidsupport reactive group and the autoinducer analogue.

[0138] In Formulae VII and VIII, above, R^(b) is either stable or it canbe cleaved by chemical or photochemical reactions. For example, R^(b)groups comprising ester or disulfide bonds can be cleaved by hydrolysisand reduction, respectively. Also within the scope of the presentinvention is the use of R^(b) groups, which are cleaved by light suchas, for example, nitrobenzyl derivatives, phenacyl groups, benzoinesters, etc. Other such cleaveable groups are well-known to those ofskill in the art.

[0139] In a preferred embodiment, the immobilized construct includes aspacer between the solid support reactive group and the autoinduceranalogue. The linker is preferably selected from C₆-C₃₀ alkyl groups,C₆-C₃₀ substituted alkyl groups, polyols, polyethers (e.g.,poly(ethyleneglycol)), polyamines, polyamino acids, polysaccharides andcombinations thereof.

[0140] In those embodiments in which the spacer moiety includes acleavable moiety, that moiety is preferably selected from groups thatare cleaved by light, heat, oxidation, reduction, enzymatic action,hydrolysis and combinations thereof, and are more preferably selectedfrom disulfides and esters.

[0141] Immunogenic Conjugates

[0142] Also provided by the present invention are immunogenic conjugateshaving a structure including a target region according to Formula IX:

[0143] In which, R¹ is preferably a member selected from —H, —OH, and(═O). R⁹ is preferably a member selected from alkyl groups andsubstituted alkyl groups. X is a member selected from —O—, —S— and —NH—.X¹ and X² are preferably members independently selected from O, S andNH. R⁹ is attached to a carrier that renders the autoinducer analogueimmunogenic or enhances the immunogenicity of the autoinducer analogue,such as a protein or adjuvant.

[0144] As used herein “target region” refers to that portion of theimmunoconjugate which an immune system component, such as an antibody, Tcell, or the like, will recognize or interact with. It is understoodthat the entire target region or only a portion thereof may interactwith an immune system component.

[0145] Commonly used carriers are large molecules that are highlyimmunogenic and capable of imparting their immunogenicity to a haptencoupled to the carrier. Examples of carriers include, but are notlimited to, proteins, lipid bilayers (e.g., liposomes), synthetic ornatural polymers (e.g., dextran, agarose, poly-L-lysine) or syntheticorganic molecules. Preferred immunogenic carriers are those that areimmunogenic, have accessible functional groups for conjugation with ahapten, are reasonably water-soluble after derivitization with a hapten,and are substantially non-toxic in vivo. Although any carrier capable ofenhancing the immunogenicity of compounds according to Formula IX, areuseful in the present invention, presently preferred carriers include,for example protein carriers having a molecular weight of greater thanor equal to 5000 daltons, more preferably, albumin or hemocyanin.

[0146] The immunogenicity of the autoinducer compositions of the presentinvention may further be enhanced by linking the autoinducer analogue toone or more peptide sequences that are able to a elicit a cellularimmune response (see, e.g., WO 94/20127). Peptides that stimulatecytotoxic T lymphocyte (CTL) responses as well as peptides thatstimulate helper T lymphocyte (HTL) responses are useful for linkage tothe compounds of the invention. The peptides can be linked by a spacermolecule. The spacer is typically comprised of relatively small, neutralmolecules, such as amino acids or amino acid mimetics, which areuncharged under physiological conditions.

[0147] A compound of the invention may be linked to a T helper peptidethat is recognized by T helper cells in the majority of the population.This can be accomplished by selecting amino acid sequences that bind tomany, most, or all of the HLA class II molecules. An example of such a Thelper peptide is tetanus toxoid at positions 830-843 (see, e.g.,Panina-Bordignon et al., Eur. J. Immunol. 19: 2237-2242 (1989)).

[0148] Further, a compound of the invention may be linked to multipleantigenic determinants to enhance immunogenicity. For example, in orderto elicit recognition by T cells of multiple HLA types, a syntheticpeptide encoding multiple overlapping T cell antigenic determinants(cluster peptides) may be used to enhance immunogenicity (see, e.g.,Ahlers et al., J. Immunol. 150: 5647-5665 (1993)). Such cluster peptidescontain overlapping, but distinct antigenic determinants. The clusterpeptide may be synthesized colinearly with a peptide of the invention.The cluster peptide may be linked to a compound of the-invention by oneor more spacer molecules.

[0149] A peptide composition comprising a compound of the inventionlinked to a cluster peptide may also be used-in conjunction with acluster peptide linked to a CTL-inducting epitope. Such compositions maybe administered via alternate routes or using different adjuvants.

[0150] Alternatively multiple peptides encoding CTL and/or HTL epitopesmay be used in conjunction with a compound of the invention.

[0151] Many methods are known to those of skill in the art for couplinga hapten to a carrier. In an exemplary embodiment, a RAA comprising asulfhydryl group is combined with keyhole limpet hemocyanin, which hasbeen activated by SMCC(succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), Dewey etal., Proc. Natl. Acad. Sci. USA 84: 5374-5378 (1987). Thesulfhydryl-bearing RAA useful in this method can be synthesized by anumber of art-recognized methods. For example, a RAA bearing a terminalcarboxyl group is coupled with cysteamine, using a dehydrating agent,such as dicyclohexylcarbodiimide (DCC), to form a dimeric RAA, linkedvia a disulfide bridge. The disulfide bridge is cleaved by reduction,affording the monomeric sulfhydryl-derivatized RAA.

[0152] In yet another preferred embodiment, R⁹ comprises a spacer moietysituated between the target component and the carrier. The discussionabove regarding the characteristics of spacer moieties located betweenthe reactive group of the solid support and the autoinducer analogue issubstantially applicable to the present embodiment. In an exemplaryembodiment, the spacer arm includes a poly(ethyleneglycol) (PEG) group.Bifunctional PEG derivative appropriate for use in this method arecommercially available (Shearwater Polymers) or can be prepared bymethods well known in the art. In an exemplary embodiment, the SMCCactivated KLH, infra, is reacted with a PEG-RAA conjugate, bearing asulfhydryl group. An appropriate conjugate can be prepared by a numberof synthetic routes accessible to those of skill in the art. Forexample, a commercially available product, such as t-Boc-NH-PEG-NH₂, isreacted with a carboxyl terminal RAA in the presence of a dehydratingagent (e.g., DCC), thereby forming the PEG amide of the RAA. The t-Bocgroup is removed by acid treatment (e.g., trifluoroacetic acid, TFA), toafford the deprotected amino PEG amide of the RAA. The deprotected RAAis subsequently reacted with a sulfhydryl protected molecule, such as3-mercaptopropionic acid or a commercially available thiol and amineprotected cysteine, in the presence of a dehydrating agent. The thiolgroup is then deprotected and the conjugate is reacted with the SMCCactivated KLH to provide an autoinducer analogue linked to a carrier viaa PEG spacer group.

[0153] The exemplary embodiments presented above are intended toillustrate general reaction schemes that are useful in preparing certainof the compounds of the present invention and should not be interpretedas limiting the scope of the invention or the pathways useful to producethe compounds of the invention.

[0154] In another preferred embodiment, the immunogenic conjugate has astructure including a target region according to Formula X:

[0155] wherein, R¹ is preferably a member selected from H, OH, and (═O);and R⁹ is preferably a member selected from alkyl and substituted alkylgroups.

[0156] In a further preferred embodiment, the target component has astructure including a target region according to Formula XI:

[0157] wherein, m is preferably a number from 0 to 30, inclusive.

[0158] Antibodies

[0159] The present invention also provides antibodies that specificallybind to autoinducer structures selected from native autoinducers (e.g.,N-(3-oxohexanoyl)homoserine lactone, N-(D-3-hydroxybutanoyl)homoserinelactone, etc.), autoinducer analogues (e.g.,N-3-(hydroxyvaleryl)homoserine lactone), immobilized autoinduceranalogues, reactive autoinducer analogues and immunogenic conjugates asdescribed herein.

[0160] “Antibody” generally refers to a polypeptide comprising aframework region from an immunoglobulin or fragments thereof thatspecifically binds and recognizes an antigen. The recognizedimmunoglobulins include the kappa, lambda, alpha, gamma, delta, epsilon,and mu constant region genes, as well as the myriad immunoglobulinvariable region genes. Light chains are classified as either kappa orlambda. Heavy chains are classified as gamma, mu, alpha, delta, orepsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA,IgD and IgE, respectively.

[0161] An exemplary immunoglobulin (antibody) structural unit comprisesa tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (VH) refer to these light and heavy chainsrespectively.

[0162] Antibodies exist, e.g., as intact immunoglobulins or as a numberof well characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′2, a dimer ofFab which itself is a light chain joined to VH-CH1 by a disulfide bond.The F(ab)′2 may be reduced under mild conditions to break the disulfidelinkage in the hinge region, thereby converting the F(ab)′2 dimer intoan Fab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3^(rd) ed. 1993).While various antibody fragments are defined in terms of the digestionof an intact antibody, one of skill will appreciate that such fragmentsmay be synthesized de novo either chemically or by using recombinant DNAmethodology. Thus, the term antibody, as used herein, also includesantibody fragments either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv).

[0163] For preparation of monoclonal or polyclonal antibodies, anytechnique known in the art can be used (see, e.g., Kohler & Milstein,Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983);Cole et al., pp. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, AlanR. Liss, Inc. (1985)).

[0164] Methods of production of polyclonal antibodies are known to thoseof skill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to the beta subunits.

[0165] When appropriately high titers of antibody to the immunogen areobtained, blood is collected from the animal and antisera are prepared.Further fractionation of the antisera to enrich for antibodies reactiveto the protein can be done if desired.

[0166] Monoclonal antibodies may be obtained by various techniquesfamiliar to those skilled in the art. Briefly, spleen cells from ananimal immunized with a desired antigen are immortalized, commonly byfusion with a myeloma cell (see Kohler & Milstein, Eur. J. Immunol. 6:511-519 (1976)). Alternative methods of immortalization includetransformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods well known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. Alternatively, one may isolate DNA sequences whichencode a monoclonal antibody or a binding fragment thereof by screeninga DNA library from human B cells according to the general protocoloutlined by Huse et al., Science 246: 1275-1281 (1989).

[0167] Monoclonal antibodies and polyclonal sera are collected andtitered against the immunogen in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Typically, polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against differentautoinducers, using a competitive binding immunoassay. Specificpolyclonal antisera and monoclonal antibodies will usually bind with aK_(d) of at least about 0.1 mM, more usually at least about 1 μM,preferably at least about 0.1 μM or better, and most preferably, 0.01 μMor better.

[0168] Techniques for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce antibodies to compounds ofthis invention and the unmodified native autoinducer molecules. Also,transgenic mice, or other organisms such as other mammals, may be usedto express humanized antibodies. Alternatively, phage display technologycan be used to identify antibodies and heteromeric Fab fragments thatspecifically bind to selected antigens (see, e.g., McCafferty et al.,Nature 348: 552-554 (1990); Marks et al., Biotechnology 10: 779-783(1992)).

[0169] In an exemplary embodiment, an animal, such as a rabbit or mouseis immunized with a RAA immunogenic construct as described above. Theantibodies produced as a result of the immunization are preferablyisolated using standard methods.

[0170] In a still further preferred embodiment, the antibody is ahumanized antibody. “Humanized” refers to a non-human polypeptidesequence that has been modified to minimize immunoreactivity in humans,typically by altering the amino acid sequence to mimic existing humansequences, without substantially altering the function of thepolypeptide sequence (see, e.g., Jones et al., Nature 321: 522-525(1986), and published UK patent application No. 8707252).

[0171] In another preferred embodiment, the present invention providesan antibody, as described above, further comprising a member selectedfrom detectable labels, biologically active agents and combinationsthereof attached to the antibody.

[0172] When the antibody is conjugated to a detectable label, the labelis preferably a member selected from the group consisting of radioactiveisotopes, fluorescent agents, fluorescent agent precursors,chromophores, enzymes and combinations thereof. Methods for conjugatingvarious groups to antibodies are well known in the art. For example, adetectable label that is frequently conjugated to an antibody is anenzyme, such as horseradish peroxidase, alkaline phosphatase,β-galactosidase, and glucose oxidase.

[0173] In an exemplary embodiment of the present invention, horseradishperoxidase is conjugated to an antibody raised against an autoinducer orautoinducer analogue. In this embodiment, the saccharide portion of thehorseradish peroxidase is oxidized by periodate and subsequently coupledto the desired immunoglobin via reductive amination of the oxidizedsaccharide hydroxyl groups with available amine groups on theimmunoglobin.

[0174] Methods of producing antibodies labeled with small molecules, forexample, fluorescent agents, are well known in the art. Fluorescentlabeled antibodies can be used in immunohistochemical staining (Osbornet al., Methods Cell Biol. 24: 97-132 (1990); in flow cytometry or cellsorting techniques (Ormerod, M. G. (ed.), FLOW CYTOMETRY. A PRACTICALAPPROACH, IRL Press, New York, 1990); for tracking and localization ofantigens, and in various double-staining methods (Kawamura, A., Jr.,FLUORESCENT ANTIBODY TECHNIQUES AND THEIR APPLICATION, Univ. TokyoPress, Baltimore, 1977).

[0175] Many reactive fluorescent labels are available commercially(e.g., Molecular Probes) or can be synthesized using art-recognizedtechniques. In an exemplary embodiment, an antibody of the invention islabeled with an amine-reactive fluorescent agent, such as fluoresceinisothiocyanate under mildly basic conditions. For other examples ofantibody labeling techniques, see, Goding, J. Immunol. Methods 13:215-226 (1976); and Goding, in, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE, pp. 6-58, Academic Press, Orlando (1988).

[0176] In another preferred embodiment, the invention provides anisolated nucleic acid encoding an antibody or a portion of an antibodyof the invention. In a further preferred embodiment, the antibodyfragment is an F_(V) fragment. F_(V) fragments of antibodies areheterodimers of antibody V_(H) (variable region of the heavy chain) andV_(L) domains (variable region of the light chain). They are thesmallest antibody fragments that contain all structural informationnecessary for specific antigen binding. F_(V) fragments are useful fordiagnostic and therapeutic applications such as imaging of tumors ortargeted cancer therapy. In particular, because of their small size,F_(V) fragments are useful in applications that require good tissue ortumor penetration, because small molecules penetrate tissues much fasterthan large molecules (Yokota et al., Cancer Res., 52: 3402-3408 (1992)).

[0177] The heterodimers of heavy and light chain domains that occur inwhole IgG, for example, are connected by a disulfide bond, but F_(V)fragments lack this connection. Although native unstabilized F_(V)heterodimers have been produced from unusual antibodies (Skerra et al.,Science, 240: 1038-1041 (1988); Webber et al., Mol. Immunol. 4: 249-258(1995), generally F_(V) fragments by themselves are unstable because theV_(H) and V_(L) domains of the heterodimer can dissociate (Glockshuberet al., Biochemistry 29: 1362-1367 (1990)). This potential dissociationresults in drastically reduced binding affinity and is often accompaniedby aggregation.

[0178] Solutions to the stabilization problem have resulted from acombination of genetic engineering and recombinant protein expressiontechniques. The most common method of stabilizing F_(V)s is the covalentconnection of V_(H) and V_(L) by a flexible peptide linker, whichresults in single chain F_(V) molecules (see, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 16: 5879-5883(1988)). The single chain F_(V)s (scF_(V)s) are generally more stablethan F_(V)s alone.

[0179] Another way to generate stable recombinant F_(V)s is to connectV_(H) and V_(L) by an interdomain disulfide bond instead of a linkerpeptide; this technique results in disulfide stabilized F_(V) (dsF_(V)).The dsF_(V)s, when they can be successfully produced, solve manyproblems that can be associated with scF_(V)s: they are very stable,often show full antigen binding activity, and sometimes have betteraffinity than scF_(V)s (Reiter et al., Int. Cancer 58: 142-149 (1994)).

[0180] Peptide linkers, such as those used in the expression ofrecombinant single chain antibodies, may be employed as the linkers andconnectors of the invention. Peptide linkers and their use arewell-known in the art. (See, e.g., Huston et al., 1988; Bird et al.,1983; U.S. Pat. No. 4,946,778; U.S. Pat. No. 5,132,405; and Stemmer et.al., Biotechniques 14:256-265 (1993)). The linkers and connectors areflexible and their sequence may vary. Preferably, the linkers andconnectors are long enough to span the distance between the amino acidsto be joined without putting strain on the structure. For example, thelinker (gly₄ser)₃ is a useful linker because it is flexible and withouta preferred structure (Freund et al., Biochemistry 33: 3296-3303(1994)).

[0181] After the stabilized immunoglobin has been designed, a geneencoding F_(V) is constructed. Methods for isolating and preparingrecombinant nucleic acids are known to those skilled in the art (see,Sambrook et al., Molecular Cloning. A Laboratory Manual (2d ed. 1989);Ausubel et al., Current Protocols in Molecular Biology (1995)).

[0182] Oligonucleotides that are not commercially available can bechemically synthesized according to the solid phase phosphoramiditetriester method first described by Beaucage & Caruthers, TetrahedronLetts. 22: 1859-1862 (1981), using an automated synthesizer, asdescribed in Van Devanter et. al., Nucleic Acids Res. 12: 6159-6168(1984). Purification of oligonucleotides is by either native acrylamidegel electrophoresis or by anion-exchange HPLC as described in Pearson &Reanier, J. Chrom. 255: 137-149 (1983).

[0183] The sequence of the cloned genes and synthetic oligonucleotidescan be verified after cloning using, e.g., the chain termination methodfor sequencing double-stranded templates of Wallace et al., Gene 16:21-26 (1981).

[0184] One preferred method for obtaining specific nucleic acidsequences combines the use of synthetic oligonucleotide primers withpoly erase extension or ligation on a mRNA or DNA template. Such amethod, e.g., RT, PCR, or LCR, amplifies the desired nucleotidesequence, which is often known (see, U.S. Pat. Nos. 4,683,195 and4,683,202). Restriction endonuclease sites can be incorporated into theprimers. Amplified polynucleotides are purified and ligated into anappropriate vector. Alterations in the natural gene sequence can beintroduced by techniques such as in vitro mutagenesis and PCR usingprimers that have been designed to incorporate appropriate mutations.

[0185] A particularly preferred method of constructing theimmunoglobulin is by overlap extension PCR. In this technique,individual fragments are first generated by PCR using primers that arecomplementary to the immunoglobulin sequences of choice. These sequencesare then joined in a specific order using a second set of primers thatare complementary to “overlap” sequences in the first set of primers,thus linking the fragments in a specified order. Other suitable F_(v)fragments can be identified by those skilled in the art.

[0186] The immunoglobulin, e.g., F_(v), is inserted into an “expressionvector,”“cloning vector,” or “vector.” Expression vectors can replicateautonomously, or they can replicate by being inserted into the genome ofthe host cell. Often, it is desirable for a vector to be usable in morethan one host cell, e.g., in E. coli for cloning and construction, andin a mammalian cell for expression. Additional elements of the vectorcan include, for example, selectable markers, e.g., tetracyclineresistance or hygromycin resistance, which permit detection and/orselection of those cells transformed with the desired polynucleotidesequences (see, e.g., U.S. Pat. No. 4,704,362). The particular vectorused to transport the genetic information into the cell is also notparticularly critical. Any suitable vector used for expression ofrecombinant proteins host cells can be used.

[0187] Expression vectors typically have an expression cassette thatcontains all the elements required for the expression of thepolynucleotide of choice in a host cell. A typical expression cassettecontains a promoter operably linked to the polynucleotide sequence ofchoice. The promoter used to direct expression of the nucleic aciddepends on the particular application, for example, the promoter may bea prokaryotic or eukaryotic promoter depending on the host cell ofchoice. The promoter is preferably positioned about the same distancefrom the heterologous transcription start site as it is from thetranscription start site in its natural setting. As is known in the art,however, some variation in this distance can be accommodated withoutloss of promoter function. Promoters include any promoter suitable fordriving the expression of a heterologous gene in a host cell, includingthose typically used in standard expression cassettes. In addition tothe promoter, the recombinant protein gene will be operably linked toappropriate expression control sequences for each host. For E. coli thisincludes a promoter such as the T7, trp, tac, lac or lambda promoters, aribosome binding site, and preferably a transcription terminationsignal. For eukaryotic cells, the control sequences will include apromoter and preferably an enhancer derived from immunoglobulin genes,SV40, cytomegalovirus, etc., and a polyadenylation sequence, and mayinclude splice donor and acceptor sequences. In one embodiment of theinvention, described in Example I, the permutated anti-Tac Fv gene isoperably linked to the T7 promoter. The T7 promoter is active inStudier's E. coli Bl21/ÿDE3 expression system (Studier & Moffatt, J.Mol. Biol. 189: 113-130 (1996)).

[0188] The vectors of the invention can be transferred into the chosenhost cell by well-known methods such as calcium chloride transformationfor E. coli and calcium phosphate treatment or electroporation formammalian cells. Cells transformed by the plasmids can be selected byresistance to antibiotics conferred by genes contained on the plasmids,such as the amp, gpt, neo and hyg genes.

[0189] Proteins of the invention can be expressed in a variety of hostcells, including E. coli, other bacterial hosts, yeast, and varioushigher eukaryotic cells such as the COS, CHO, and HeLa cells lines andmyeloma cell lines. Methods for refolding single chain polypeptidesexpressed in bacteria such as E. coli have been described and arewell-known and are applicable to the polypeptides of this invention.(See, e.g., Buchner et al., Analytical Biochemistry 205: 263-270 (1992);Pluckthun, Biotechnology 9: 545 (1991); Huse, et al., Science 246: 1275(1989) and Ward et al., Nature 341: 544 (1989)).

[0190] Often, functional protein from E. coli or other bacteria isgenerated from inclusion bodies and requires the solubilization of theprotein using strong denaturants, and subsequent refolding. In thesolubilization step, a reducing agent must be present to dissolvedisulfide bonds as is well-known in the art. Renaturation to anappropriate folded form is typically accomplished by dilution (e.g.100-fold) of the denatured and reduced protein into refolding buffer.

[0191] Once expressed, the recombinant proteins can be purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, and the like(see, generally, Scopes, PROTEIN PURIFICATION (1982)). Substantiallypure compositions of at least about 90 to 95% homogeneity are preferred,and 98 to 99% or more homogeneity are most preferred for pharmaceuticaluses. Once purified, partially or to homogeneity as desired, thepolypeptides may then be used therapeutically and diagnostically.

[0192] Targeted Bioactive Agents

[0193] In another preferred embodiment, the antibodies and RAA of theinvention further include a biologically active agent, preferably anantibiotic. Methods similar to those discussed above in the context offluorescent labels can be used to attach various antibiotics to anantibody of the invention. An array of methods for attachingbiologically active agents, such as antibiotics, to an antibody areavailable to those of skill in the art. Exemplary antibiotic compoundsfor conjugation to an antibody or RAA of the invention are set forth inTable 2 TABLE 2 Penicillins¹ penicillin G, amoxicillin, nafcillinampicillin, ticarcillin, negative carbenicillin, cloxacillin, penicillinV, piperacillin Cephalosporins² cefoxitin, ceforanide polymyxinpolymyxin B, colistin, cefepime,3-thiaol-4-yl-carba-1-dethiacephalosporin Vancomycin³ daptomycin,vancomycin teicoplanin, ristocetin Biosurfactants⁴ circulin, EM49,polypeptin, brecistin, cerexin, tridecephin, surfactin, subsporin,mycosubtilisin, bacillomycin Miscellaneous Antibiotics⁵ capreomycin,bacitracin, gramicidin, gramicidin S, tyrocidine, tazobactam, imipenem,piperacillin-tazobactam, ciprofloxacin, ceftriaxone, ceftazidimeAmantadine⁶ Polyene macrolide⁷ amphotericin Endotoxin bindingtachyplesin⁸ LPS-binding antiendotoxin factor⁹ LPS binding proteinLPS-binding anti-endotoxin (human)¹⁰

[0194] Numerous agents have been developed for the cross-linking ofbiological molecules (see, for example, Pierce Chemical Co., (Rockford,Ill.), General Catalog, pp. E-10-E-39 (1992)). In general, cross-linkingagents possess functional groups that are reactive with the side chainsof different amino acids found in proteins or peptides. Variousfunctional groups will react with primary amino groups, carboxyl groups,hydroxyl groups, or thiol groups of proteins (e.g., antibodies) or othercompounds (e.g., RAA). In the design of antibody or RAA-antibioticconjugates, the reactive groups of both of the components must beconsidered. In general, antibodies have many reactive groups that can beused in direct conjugation schemes (amino acids containing primaryamine, carboxyl; hydroxyl, thiol (after reduction)) or modified groups(glycosylated amino acids that can be oxidized to aldehyde; or primaryamines that can be made thiol-reactive) for conjugation schemes.Similarly, the RAA of the invention can be prepared with carefullyselected reactive functional groups. The selection of an antibiotic froma family of related compounds and the selection of a cross-linkingscheme must also take into consideration the reactive groups on anantibiotic. Methods of forming antibody conjugates are well known in theart (see, for example, Shikhani et al. U.S. Pat. No. 5,998,381).

[0195] In an exemplary embodiment, the antibiotic attached to theantibody or RAA is an aminoglycoside. Aminoglycosides are all potentbactericidal agents that share the same general range of antibacterialactivity and pharmacokinetic behavior. The members of the group aretypified by the presence of aminosugars glycosidically linked toaminocyclitols. The main agents fall into two groups: the small groupconsisting of streptomycin, and its close relatives; and the large groupwhich is subdivided into the neomycin group, the kanamycin group whichis again subdivided into the kanamycins, tobramycin and theirsemi-synthetic derivatives amikacin and dibekacin and the importantsub-group of gentamicins and their relatives, netilmicin and sissomicin.

[0196] Each of the aminoglycosides has a amino group that can bederivatized to tether the antibiotic to an antibody or RAA. For example,the amine group of an amino glycoside can be converted to a carboxylgroup by reacting the antibiotic with an anhydride, such as succinicanhydride. The succinyl antibiotic is isolated and purified, if desired.The free carboxyl group of the succinyl moiety is then reacted with anamine on the antibody or RAA, typically in the presence of awater-soluble dehydrating reagent, such as1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDCI(Aldrich), to form an amide linkage between the carboxyl group and theantibody amine.

[0197] In another exemplary embodiment, the linker between the antibodyor RAA and the antibiotic includes a cleavable moiety, such as thecis-aconityl group. The cis-aconityl group is derived from cis-aconiticanhydride (Aldrich) and the antibody-antibiotic conjugate is assembledsubstantially as described above for the succinyl derivative. (Shen etal., Biochem. Biophys. Res. Commun. 102: 1048 (1981). The cis-aconitylmoiety is stable at neutral pH, but rapidly cleaves at a pH below about4.5, such as might be found in an endocytotic vacuole.

[0198] Many other combinations of bioactive agents, linker moieties andmethods of attaching the bioactive agent to the antibiotic will beapparent to those of skill in the art. The above discussion is intendedto be illustrative of the invention and it should not be interpreted aslimiting the scope of the invention or the claims as set forth herein.

[0199] Pharmaceutical Formulations

[0200] In another preferred embodiment, the present invention provides apharmaceutical formulation comprising the immunogenic conjugateincluding the target structure according to Formula IX and apharmaceutically acceptable carrier.

[0201] In a still further preferred embodiment, the invention provides apharmaceutical formulation including a pharmaceutically acceptablecarrier and a conjugate of a bioactive agent with an antibody or RAA ofthe invention.

[0202] The compounds described herein, or pharmaceutically acceptableaddition salts or hydrates thereof, can be delivered to a patient usinga wide variety of routes or modes of administration. Suitable routes ofadministration include, but are not limited to, inhalation, transdermal,oral, rectal, transmucosal, intestinal and parenteral administration,including intramuscular, subcutaneous and intravenous injections.

[0203] The compounds described herein, or pharmaceutically acceptablesalts and/or hydrates thereof, may be administered singly, incombination with other compounds of the invention, and/or in cocktailscombined with other therapeutic agents. Of course, the choice oftherapeutic agents that can be co-administered with the compounds of theinvention will depend, in part, on the condition being treated.

[0204] For example, when administered to patients suffering from adisease state caused by an organism that relies on an autoinducer, thecompounds of the invention can be administered in cocktails containingagents used to treat the pain, infection and other symptoms and sideeffects commonly associated with the disease. Such agents include, e.g.,analgesics, antibiotics, etc.

[0205] When administered to a patient undergoing cancer treatment, thecompounds may be administered in cocktails containing anti-cancer agentsand/or supplementary potentiating agents. The compounds may also beadministered in cocktails containing agents that treat the side-effectsof radiation therapy, such as anti-emetics, radiation protectants, etc.

[0206] Supplementary potentiating agents that can be co-administeredwith the compounds of the invention include, e.g., tricyclicanti-depressant drugs (e.g., imipramine, desipramine, arnitriptyline,clomipramine, trimipramine, doxepin, nortriptyline, protriptyline,amoxapine and maprotiline); non-tricyclic and anti-depressant drugs(e.g., sertraline, trazodone and citalopram); Ca⁺² antagonists (e.g.,verapamil, nifedipine, nitrendipine and caroverine); amphotericin;triparanol analogues (e.g., tamoxifen); antiarrhythmic drugs (e.g.,quinidine); antihypertensive drugs (e.g., reserpine); thiol depleters(e.g., buthionine and sulfoximine); and calcium leucovorin.

[0207] The active compound(s) of the invention are administered per seor in the form of a pharmaceutical composition wherein the activecompound(s) is in admixture with one or more pharmaceutically:acceptable carriers, excipients or diluents. Pharmaceutical compositionsfor use in accordance with the present invention are typicallyformulated in a conventional manner using one or more physiologicallyacceptable carriers comprising excipients and auxiliaries, whichfacilitate processing of the active compounds into preparations' which,can be used pharmaceutically. Proper formulation is dependent upon theroute of administration chosen.

[0208] For injection, the agents of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks's solution, Ringer's solution, or physiological saline buffer.For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

[0209] For oral administration, the compounds can be formulated readilyby combining the active compound(s) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxyniethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

[0210] Dragee cores are provided with suitable coatings. For thispurpose, concentrated sugar solutions may be used, which may optionallycontain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,polyethylene glycol, and/or titanium dioxide, lacquer solutions, andsuitable organic solvents or solvent mixtures. Dyestuffs or pigments maybe added to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

[0211] Pharmaceutical preparations, which can be used orally, includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

[0212] For buccal administration, the compositions may take the form oftablets or lozenges formulated in conventional manner.

[0213] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

[0214] The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents may be added, such as thecross-linked polyvinyl pyrrolidorie, agar, or alginic acid or a saltthereof such as sodium alginate.

[0215] Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances, which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents, which increase the solubility of thecompounds to allow for the preparation of highly, concentratedsolutions.

[0216] Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

[0217] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0218] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation ortranscutaneous delivery (e.g., subcutaneously or intramuscularly),intramuscular injection or a transdermal patch. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (e.g., as an emulsion in an acceptable oil) or ion exchangeresins, or as sparingly soluble derivatives, for example, as a sparinglysoluble salt.

[0219] The pharmaceutical compositions also may comprise suitable solidor gel phase carriers or excipients. Examples of such carriers orexcipients include but are not limited to calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols.

[0220] Vaccines

[0221] In another preferred embodiment, the present invention provides avaccine effective for preventing or reducing microbial infection in asubject to whom the vaccine is administered. The vaccine will preferablyinclude the immunogenic conjugate, including the target region accordingto Formula IX, although unconjugated autoinducers and autoinduceranalogues are also useful in this embodiment. The vaccines of theinvention can produce a humoral response, a cellular response or acombination of these two responses.

[0222] The vaccines of the present invention are preferablyepitope-based vaccines, wherein at least a portion of the molecularstructure of an autoinducer or autoinducer analogue serves as theepitope, or antigenic determinant. In the interest of clarity, thediscussion below focuses on the use of an autoinducer. It is to beunderstood that the term autoinducer also encompasses autoinduceranalogues, immunogenic conjugates of autoinducers and combinations ofthese compositions.

[0223] Evidence has demonstrated the value of an epitope approach todisease treatment. More specifically, vaccination, with either dominantor subdominant epitopes has been shown to be useful in combatingparasitic, microbial infections (Le et al., Vaccine 16: 305 (1998);Wanget al., J. Immunol. 157: 4061 (1996); and Franke et al., J. Immunol.159: 424, (1997). Other studies have demonstrated that epitope vaccinesare protective against acute or chronic viral infection in systems suchas influenza or LCMV infection (Oukka et al., J. Immunol. 157: 3039(1996); Tourdot et al., J. Immunol. 159: 2391 (1997); van der Most etal., J. Immunol. 157: 5543 (1996); van der Most et al., J. Virol. 71:5110 (1997); An et al., J. Virol. 71: 2292 (1997)).

[0224] Furthermore, a variety of assays to detect and quantify theaffinity of interaction between an epitope and MHC have also beenestablished (Sette et. al., Curr. Opin. Immunol. 4: 79 (1992);Sinigaglia et al., Curr. Biol. 6: 52 (1994); Engelhard, Curr. Opin.Immunol. 6: 13 (1994). Finally, a threshold of affinity associated withgeneration of an immune response has also been elucidated (Schaeffer etal., Proc. Natl. Acad. Sci. USA 86: 4649 (1989). Thus, by a combinationof motif searches and MHC-epitope binding assays, potential candidatesfor epitope-based vaccines can be identified.

[0225] Various strategies can be utilized to evaluate immunogenicity andto identify immunogenic epitopes, including:

[0226] 1) Evaluation of primary T cell cultures from normal individuals(see, e.g., Wentworth et al., Mol. Immunol. 32: 603 (1995); Celis etal., Proc. Natl. Acad. Sci. USA 91: 2105 (1994); Tsai et al., J.Immunol. 158: 1796 (1997); Kawashima et al., Human Immunol. 59: 1(1998)) The procedure typically involves the stimulation of peripheralblood lymphocytes (PBL) from normal subjects with a test autoinducer inthe presence of antigen presenting cells in vitro over a period ofseveral weeks. T cells specific for the autoinducer become activatedduring this time and are detected using, e.g., a ⁵¹Cr-release assayinvolving autoinducer-sensitized target cells.

[0227] 2) Immunization of HLA transgenic mice (see, e.g., Wentworth etal., J. Immunol. 26: 97 (1996); Wentworth et al., Int. Immunol. 8: 651(1996); Alexander et al., J. Immunol. 159: 4753 (1997)). In this method,autoinducer in incomplete Freund's adjuvant is administeredsubcutaneously to HLA transgenic mice. Several weeks followingimmunization, splenocytes are removed and cultured in vitro in thepresence of test autoinducer for approximately one week.Autoinducer-specific T cells are detected using, e.g., a ⁵¹Cr-releaseassay involving autoinducer sensitized target cells and target cellsexpressing endogenously generated antigen.

[0228] 3) Demonstration of recall T cell responses from patients whohave been effectively vaccinated or who have a tumor; (see, e.g.,Rehermann et al., J. Exp. Med. 181: 1047 (1995); Doolan et al., Immunity7: 97 (1997); Bertoni et al., J. Clin. Invest. 100: 503 (1997);Threlkeld et al., J. Immunol. 159: 1648 (1997); Diepolder et al., J.Virol. 71: 6011 (1997); Tsang et al., J. Natl. Cancer Inst. 87-982(1995); Disis et al., J. Immunol. 156: 3151 (1996)). In applying thisstrategy, recall responses are detected by culturing PBL from patientswith a disease who have generated an immune response “naturally”, orfrom patients who were vaccinated with autoinducer antigen vaccines. PBLfrom subjects are cultured in vitro for 1-2 weeks in the presence oftest autoinducer plus antigen presenting cells (APC) to allow activationof “memory” T cells, as compared to “naive” T cells. At the end of theculture period, T cell activity is detected using assays for T cellactivity including ⁵¹Cr release involving autoinducer-sensitizedtargets, T cell proliferation, or lymphokine release.

[0229] Conventional assays utilized to detect T cell responses includeproliferation assays, lymphokine secretion assays, direct cytotoxicityassays, and limiting dilution assays. For example, antigen-presentingcells that have been incubated with a peptide can be assayed for theability to induce CTL responses in responder cell populations.Antigen-presenting cells can be normal cells such as peripheral bloodmononuclear cells or dendritic cells. Alternatively, mutant non-humanmammalian cell lines that are deficient in their ability to load class Imolecules with internally processed peptides and that have beentransfected with the appropriate human class I gene, may be used to testfor the capacity of the peptide to induce in vitro primary CTLresponses.

[0230] Peripheral blood mononuclear cells (PBMCs) may be used as theresponder cell source of CTL precursors. The appropriateantigen-presenting cells are incubated with peptide, after which thepeptide-loaded antigen-presenting cells are then incubated with theresponder cell population under optimized culture conditions. PositiveCTL activation can be determined by assaying the culture for thepresence of CTLs that kill radio-labeled target cells.

[0231] More recently, a method has been devised which allows directquantification of antigen-specific T cells by staining withfluorescein-labeled HLA tetrameric complexes (Altman et al., Proc. Nat.Acad. Sci. USA 90: 10330 (1993); Altman et al., Science 214: 94 (1996)).Other relatively recent technical developments include staining forintracellular lymphokines, and interferon-γ release assays or ELISPOTassays. Tetramer staining, intracellular lymphokine staining and ELISPOTassays all appear to be at least 10-fold more sensitive than moreconventional assays (Lalvani et al., J. Exp. Med. 186: 859 (1997);Dunbar et al., Curr. Biol. 8: 413 (1998); Murali-Krishna et al.,Immunity 8: 177 (1998)).

[0232] Once appropriately immunogenic epitopes have been defined, theycan be sorted and delivered by various means, including lipopeptides(Vitiello et al., J. Clin. Invest. 95: 341 (1995)), viral deliveryvectors (Perkus et al., in, CONCEPTS IN VACCINE DEVELOPMENT, Kaufmann(ed.), p. 379, 1996; Chakrabarti et al., Nature 320: 535 (1986); Hu etal., Nature 320: 537 (1986); Kieny et al., AIDS Bio/Technology 4: 790(1986); Top et al., J. Infect. Dis. 124: 148 (1971); Chanda et al.,Virology 175:535 (1990)), particles of viral or synthetic origin (Kofleret al., J. Immunol. Methods. 192: 25 (1996); Eldridge et al., Sem.Hematol. 30: 16 (1993); Falo et al., Nature Med. 7: 649 (1995)),adjuvants (Warren et al., Annu. Rev. Immunol. 4: 369 (1986); Gupta etal., Vaccine 11: 293 (1993)) and liposomes (Reddy et al., J. Immunol.148: 1585 (1992); Rock, Immunol. Today 17: 131 (1996)).

[0233] Immunogenic compositions suitable for use as vaccines may beprepared from immunogenic conjugates as disclosed herein. Theimmunogenic conjugate elicits an immune response, which producesantibodies that bind to autoinducers, thereby inactivating them.

[0234] Vaccines may be prepared as injectables, as liquid solutions oremulsions. The peptides may be mixed with pharmaceutically-acceptableexcipients, which are compatible with the peptides. Excipients mayinclude water, saline, dextrose, glycerol, ethanol, and combinationsthereof. The vaccine may further contain auxiliary substances such aswetting or emulsifying agents, pH buffering agents, or adjuvants toenhance the effectiveness of the vaccines.

[0235] The immunogens of this invention may be combined or mixed withvarious solutions and other compounds as is known in the art. Forexample, an immunogen may be administered in water, saline or bufferedvehicles with or without various adjuvants or immunodiluting agents.Examples of such adjuvants or agents include aluminum hydroxide,aluminum phosphate, aluminum potassium sulfate (alum), berylliumsulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-wateremulsions, muramyl dipeptide, bacterial endotoxin, lipid X,Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis,polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A,saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers orother synthetic adjuvants. Such adjuvants are available commerciallyfrom various sources, for example, Merck Adjuvant 65 (Merck and Company,Inc., Rahway, N.J.) or Freund's Incomplete Adjuvant and CompleteAdjuvant (Difco Laboratories, Detroit, Mich.). Other suitable adjuvantsare Amphigen (oil-in-water), Alhydrogel (aluminum hydroxide), or amixture of Amphigen and Alhydrogel. Only aluminum is approved for humanuse. The discussion above concerning pharmaceutical formulations is alsorelevant to the formulation of the vaccines of the invention.

[0236] Preferably, the vaccines are formulated to contain a finalconcentration of immunogen in the range of from 0.2 to 200 μg/ml,preferably-5 to 50 μg/ml, most preferably 15 μg/ml. After formulation,the vaccine may be incorporated into a sterile container, which is thensealed and stored at a low temperature, for example 4° C., or it may befreeze-dried. Lyophilization permits long-term storage in a stabilizedform.

[0237] Administration

[0238] The vaccines may be administered by any conventional methodsincluding oral administration and parenteral (e.g., subcutaneous orintramuscular) injection. The treatment can consist of a single dose ofvaccine or a plurality of doses over a period of time. The immunogen ofthe invention can be combined with appropriate doses of compoundsincluding other epitopes of the target bacteria. Also, the immunogen canbe a component of a recombinant vaccine, which could be adaptable fororal administration.

[0239] The recipient of the vaccine is preferably a mammal. Although usein humans in preferred, veterinary use of the compositions of theinvention is also contemplated

[0240] The proportion of immunogen and adjuvant can be varied over abroad range so long as both are present in effective amounts. Forexample, aluminum hydroxide can be present in an amount of about 0.5% ofthe vaccine mixture (Al₂O₃ basis). On a per-dose basis, the amount ofthe immunogen can range from about 5 μg to about 100 μg of immunogen perpatient of about 70 kg. A preferable range is from about 20 μg to about40 μg per dose. A suitable dose size is about 0.5 ml. Accordingly, adose for intramuscular injection, for example, would comprise 0.5 mlcontaining 20 μg of immunogen in admixture with 0.5% aluminum hydroxide.

[0241] Vaccines of the invention may be combined with other vaccines forother diseases to produce multivalent vaccines. A pharmaceuticallyeffective amount of the immunogen can be employed with apharmaceutically acceptable carrier such as a protein or diluent usefulfor the vaccination of mammals, particularly humans. Other vaccines maybe prepared according to methods well-known to those skilled in the art.

[0242] The vaccines of the present invention may also be administered inconjunction with immune stimulating complexes (ISCOMs). ISCOMs arenegatively charged cage-like structure of 30-40 nm in size formedspontaneously on mixing cholesterol and Quil A (saponin). Protectiveimmunity has been generated in a variety of experimental models ofinfection including toxoplasmosis and Epstein-Barr virus-induced tumorsusing ISCOMs as the delivery vehicle for antigens (see, e.g., Mowat etal., Immunol. Today, 23: 383-385 (1991)). Immunogenic compositions usingISCOMs are comprised of a compound or composition of the inventionencapsulated into ISCOMs for delivery.

[0243] Immunotherapy regimens which produce maximal immune responsesfollowing the administration of the fewest number of doses, ideally onlyone dose, are highly desirable. This result can be approached throughentrapment of immunogen in macromolecular vehicles, such asmicroparticles, liposomes and the like. For example, the absorbablesuture material poly(lactide-co-glycolide) co-polymer can be fashionedinto microparticles containing immunogen (see, e.g., Eldridge et al.,Molec. Immunol., 28: 287-294 (1991); Moore et al., Vaccine 13: 1741-1749(1995); and Men et al., Vaccine, 13: 683-689 (1995)). Following oral orparenteral administration, microparticle hydrolysis in vivo produces thenon-toxic byproducts, lactic and glycolic acids, and releases immunogenlargely unaltered by the entrapment process. Microparticle formulationscan also provide primary and subsequent booster immunizations in asingle administration by mixing immunogen entrapped microparticles withdifferent release rates. Single dose formulations capable of releasingantigen ranging from less than one week to greater than six months canbe readily achieved.

[0244] The immunogens of the invention may also be administered vialiposomes, which serve to target the immunogens to a particular tissue,such as lymphoid tissue, or targeted selectively to infected cells, aswell as increase the half-life of the immunogen composition. Liposomesinclude emulsions, foams, micelles, insoluble monolayers, liquidcrystals, phospholipid dispersions, lamellar layers and the like. Inthese preparations the immunogen to be delivered is incorporated as partof a liposome, alone or in conjunction with a molecule which binds to,e.g., a receptor prevalent among lymphoid cells, such as monoclonalantibodies which bind to the CD45 antigen, or with other therapeutic orimmunogenic compositions. Thus, liposomes either filled or decoratedwith a desired immunogen of the invention can be directed to the site oflymphoid cells, where the liposomes then deliver the immunogencompositions. Liposomes for use in the invention are formed fromstandard vesicle-forming lipids, which generally include neutral andnegatively charged phospholipids and a sterol, such as cholesterol. Theselection of lipids is generally guided by consideration of, e.g.,liposome size, acid lability and stability of the liposomes in the bloodstream. A variety of methods are available for preparing liposomes, asdescribed in, e.g., Szoka, et al., Ann. Rev. Biophys. Bioeng., 9: 467(1980), and U.S. Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and5,019,369.

[0245] Libraries

[0246] Also within the scope of the present invention are libraries ofthe autoinducer analogues, immunogenic conjugates and antibodies of theinvention. The libraries will preferably include at least 10 compounds,more preferably at least 100 compound, even more preferably at least1000 compounds and still more preferably at least 100,000 compounds.

[0247] Parallel, or combinatorial, synthesis has as its primaryobjective the generation of a library of diverse molecules which allshare a common feature, referred to throughout this description as ascaffold. By substituting different moieties at each of the variableparts of the scaffold molecule, the amount of space explorable in alibrary grows. Theories and modem medicinal chemistry advocate theconcept of occupied space as a key factor in determining the efficacy ofa given compound against a given biological target. By creating adiverse library of molecules, which explores a large percentage of thetargeted space, the odds of developing a highly efficacious leadcompound increase dramatically.

[0248] Parallel synthesis is generally conducted on a solid phasesupport, such as a polymeric resin. The scaffold, or other suitableintermediate is cleavably tethered to the resin by a chemical linker.Reactions are carried out to modify the scaffold while tethered to theparticle. Variations in reagents and/or reaction conditions produce thestructural diversity, which is the hallmark of each library.

[0249] Parallel synthesis of “small” molecules (non-oligomers with amolecular weight of 200-1000) was rarely attempted prior to 1990. See,for example, Camps. et al., Annaks de Quimica, 70: 848-(1990). Recently,Ellmann disclosed the solid phase-supported parallel (also referred toas “combinatorial”) synthesis of eleven benzodiazepine analogs alongwith some prostaglandins and beta-turn mimetics. These disclosures areexemplified in U.S. Pat. No. 5,288,514. Another relevant disclosure ofparallel synthesis of small molecules may be found in U.S. Pat. No.5,324,483. This patent discloses the parallel synthesis of between 4 and40 compounds in each of sixteen different scaffolds. Chen et al. havealso applied organic synthetic strategies to develop non-peptidelibraries synthesized using multi-step processes on a polymer support.(Chen et al., J. Am. Chem. Soc., 1.16: 2661-2662 (1994)).

[0250] In an exemplary embodiment of this aspect of the invention,referring to the synthesis of compound 4, set forth above, thecarboxylic acid-derived portion (“arm”) of an autoinducer library istethered via one of the two available carboxyl groups to a solidsupport, such as an amine-containing resin. For a library of at leastten compounds, this step is repeated with nine more portions of resin,each time using an arm having a structure that is different from that ofany of the other arms. In the simplest embodiment, in which the libraryincludes 10 compounds, each of the pools of resin having a different armis reacted with the same heterocyclic moiety, thereby producing alibrary of 10 compounds that has diversity in the structure of the arms.

[0251] In a more complex library, each of the 10 pools of resin, above,is divided into, for example, 10 subpools and each of the 10 subpoolshaving a common arm is reacted with a different heterocyclic moiety,producing a single unique compound per subpool, 10 unique compounds perpool and a library having diversity in both the arms and the heterocycleand including 100 unique compounds. Many methods of introducingmolecular structural diversity into libraries of compounds are known inthe art and are appropriate for use in the present invention (see, forexample, COMBINATORIAL CHEMISTRY AND MOLECULAR DIVERSITY IN DRUGDISCOVERY, Gordon et al. (eds.), Wiley-Liss, New York, 1998.

[0252] Once a library of unique compounds is prepared, the preparationof a library of immunoconjugates, or antibodies can be prepared usingthe library of autoinducers as a starting point and using the methodsdescribed herein.

[0253] Kits

[0254] In another aspect, the present invention provides kits containingone or more of the compounds or compositions of the invention anddirections for using the compound or composition. In a preferredembodiment, the invention provides a kit for detecting an autoinducer ina sample. The kit includes an antibody that binds specifically to theautoinducer and directions for using the antibody to detect theautoinducer. Other formats for kits will be apparent to those of skillin the art and are within the scope of the present invention.

[0255] Methods

[0256] In addition to the compositions and constructs described above,the present invention also provides a number of methods that can bepracticed utilizing the compounds and conjugates of the invention.

[0257] Purification.

[0258] In another preferred embodiment, the present invention provides amethod for isolating a microbial receptor, which binds to a moleculehaving as a portion of its structure the group according to Formula I.The method preferably comprises, contacting a microbial preparation thatincludes the receptor with an immobilized compound according Formula IX,thereby forming a complex between the receptor and the immobilizedcompound.

[0259] The method of the invention for isolating a microbial receptorwill typically utilize one or more affinity chromatography techniques.Affinity chromatography enables the efficient isolation of species suchas biological molecules or biopolymers by utilizing their recognitionsites for certain supported chemical structures with a high degree ofselectivity. The literature is replete with articles, monographs, andbooks on the subject of affinity chromatography, including such topicsas affinity chromatography supports, crosslinking members, ligands andtheir preparation and use. A sampling of those references includes:Ostrove, Methods Enzymol. 182: 357-71 (1990); Ferment, Bioeng. 70:199-209 (1990). Huang et al., J. Chromatogr. 492: 431-69 (1989);“Purification of enzymes by heparin-Sepharose affinity chromatography,”J. Chromatogr., 184: 335-45 (1980); Farooqi, Enzyme Eng., 4: 441-2(1978); Nishikawa, Chem. Technol., 5(9): 564-71 (1975); Guilford et al.,in, PRACT. HIGH PERFORM. LIQ. CHROMATOGR., Simpson (ed.), 193-206(1976); Nishikawa, Proc. Int. Workshop Technol. Protein Sep. Improv.Blood Plasma Fractionation, Sandberg (ed.), 422-35; (11977) “Affinitychromatography of enzymes,” Affinity Chromatogr., Proc. Int. Symp.25-38, (1977) (Pub. 1978); and AFFINITY CHROMATOGRAPHY: A PRACTICALAPPROACH, Dean et al. (ed.), IRL Press Limited, Oxford Englarid (1985).Those of skill in the art will have ample guidance in developingparticular affinity chromatographic methods utilizing the materials ofthe invention.

[0260] In the present method, affinity chromatographic media of varyingchemical structures can be used as supports. For example, agarose gelsand cross-linked agarose gels are useful as support materials, becausetheir hydrophilicity makes them relatively free of nonspecific binding.Other useful supports include, for example, controlled-pore glass (CPG)beads, cellulose particles, polyacrylamide gel beads and Sephadex™ gelbeads' made from dextran and epichlorohydrin.

[0261] The starting point for an affinity separation is the reversibleinteraction between a receptor (R) and a ligand for that receptor (L) asexpressed by the following equation:

R(receptor)+L(ligand)=RL complex.

[0262] In affinity chromatography, this reaction between the receptorand the affinity matrix-bound ligand (e.g., autoinducer analogue) isused to selectively extract the receptor from crude solutions, either asa batch or continuous process. After washing away the contaminatingproteins, the receptor is freed from the matrix by introducing freeligand, which competes with the matrix for binding to the receptor. Theeluted receptor is freed of inhibitor by dialysis or ultrafiltrationtechniques, or in the case of strong binding ligands by chemicalprocesses that interfere with binding of the ligand to the receptor.

[0263] The affinity matrix is typically packed in a cylindrical columnto permit a continuous flow process, although batch operation are areasonable alternative for extracting the enzyme from crudepreparations. The affinity column can be protected by a guard column ofnative supporting matrix (e.g. Sepharose CL-4B) to filter out proteinsthat bind non specifically to the matrix and would be difficult to washaway completely from the bound receptor-ligand complex.

[0264] Both columns are typically equilibrated with a buffer at neutralpH prior to application of the receptor solution, for which 3 or morecolumn volumes is regarded as sufficient. Any nonreactive compound witha pKa near neutrality is satisfactory. Quite often a buffer such asN-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES) provesto be satisfactory. Other components can be included in the buffer, suchas sucrose, 3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate(CHAPS) to slow the rate of dissociation of, for example, receptorshaving multiple subunits. Other useful buffer solutions and additivesfor a given purpose will be apparent to those of skill in the art.

[0265] The flow rate through the column can vary over a wide range. Animportant consideration in the choice of flow rates is the rate constantfor association and dissociation of the receptor and the ligand. Whenthe rates of association are slower than normal, slow flow rates aregenerally used (e.g., flow rate equivalent to 1/4 bed volume per hour)

[0266] Following its immobilization on the affinity matrix, the boundreceptor is generally washed with buffer to remove the last traces ofcontaminating proteins. Extensive washing, using as much as 20 columnvolumes of buffer, is desirable and generally leads to no detectableloss during this process.

[0267] Following the purification, it is often be desirable to removethe purified molecule (e.g., receptor) from the chromatographic matrix.Thus, in a preferred embodiment, the method of the invention furthercomprises disrupting the complex between the purified molecule and themolecule, thereby separating the molecule from the particle. Thereceptor is typically eluted from the matrix by including the freeligand, or another compound that binds the receptor in the wash buffer.Again, the rate constants for equilibration with particular ligandsrequire special consideration. The half-life for dissociation of theligand can be used as a guide for ascertaining appropriate flow rates.If the rates are too slow to use continuous flow to elute the receptor,elution is accomplished in a batch process, in which the column isflooded with free ligand and then incubated for a period (e.g.,overnight) to allow the exchange to reach equilibrium before restartingthe flow to elute the receptor.

[0268] Lastly, purified receptor can be separated from the RL complex.At this point, the purified receptor is complexed with the elutingligand. Removal of the ligand is generally accomplished by dialysis ordiafiltration. Similar methods are available for purification of otherbiomolecules.

[0269] In a variation on this method, the receptors can be utilized ascomponents of the affinity matrix to isolate the components to whichthey bind.

[0270] Although the above discussion focuses on the purification ofcellular receptors, it is within the scope of the present invention toutilize the affinity chromatographic method for the purification of anymember of any class of molecules, including, for example, peptides,ligands, enzymes, enzyme substrates, carbohydrates, nucleic acids,antibodies, antigens and combinations thereof.

[0271] In a variation on the above-described method, the invention alsoprovides a method of isolating an autoinducer. The method includes thesteps of: (a) providing a sample comprising the autoinducer; (b)contacting the sample with an antibody that specifically binds to theautoinducer, thereby forming an antibody-autoinducer complex; and (c)isolating the autoinducer by isolating the antibody-autoinducer complex.The preferred embodiments of this method are substantially similar tothose discussed above, with the exception that the antibody, and not theautoinducer analogue, is preferably bound to a support.

[0272] Assays

[0273] The compositions and compounds of the invention can also be usedin an array of different assay formats. In the interest of clarity, theuse of the compositions and compounds in assays is illustrated byrelying on immunoassays as a representative assay motif. It will beapparent to those of skill in the art that the compositions of theinvention are applicable to assay formats other than immunoassays.

[0274] A variety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular autoinducer. For example,solid-phase ELISA immunoassays are routinely used to select antibodiesspecifically immunoreactive with a protein or other substance (see,e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity). Typically a specific or selectivereaction will be at least twice background signal or noise and moretypically more than 10 to 100 times background.

[0275] In addition to the detection of autoinducers, immunoassays can beused to qualitatively or quantitatively analyze autoinducers. A generaloverview of the applicable technology can be found in Harlow & Lane,supra.

[0276] Methods of producing polyclonal and monoclonal antibodies thatreact specifically with autoinducers are known to those of skill in theart (see, e.g., Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY (1991); Harlow& Lane, supra; Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE(2d ed. 1986); and Kohler & Milstein, Nature 256: 495-497 (1975). Suchtechniques include antibody preparation by, for example, selection ofantibodies from libraries of recombinant antibodies in phage or similarvectors, as well as preparation of polyclonal and monoclonal antibodiesby immunizing rabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989); Ward et al., Nature 341: 544-546 (1989)).

[0277] Once the specific antibodies against a an autoinducer areavailable, the autoinducer can be detected by a variety of immunoassaymethods. For a review of immunological and immunoassay procedures, seeBasic and Clinical Immunology (Stites & Terr eds., 7^(th) ed. 1991).Moreover, the immunoassays of the present invention can be performed inany of several configurations, which are reviewed extensively in EnzymeImmunoassay (Maggio, ed., 1980); and Harlow & Lane, supra.

[0278] Autoinducers can be detected and/or quantified using any of anumber of well recognized immunological binding assays (see, e.g., U.S.Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a reviewof the general immunoassays, see also Methods in Cell Biology:Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic andClinical Immunology (Stites & Terr, eds., 7^(th) ed. 1991).Immunological binding assays (or immunoassays) typically use an antibodythat specifically binds to a protein or antigen of choice (in this casean autoinducer or a substructure thereof). The antibody (e.g.,anti-autoinducer) may be produced by any of a number of means well knownto those of skill in the art and as described above.

[0279] Immunoassays also often use a labeling agent to specifically bindto and label the complex formed by the antibody and antigen. Thelabeling agent may itself be one of the moieties comprising theantibody/antigen complex. Thus, the labeling agent can be a labeledautoinducer or a labeled anti-autoinducer antibody. Alternatively, thelabeling agent may be a third moiety, such as a secondary antibody, thatspecifically binds to the antibody/autoinducer complex (a secondaryantibody is typically specific to antibodies of the species from whichthe first antibody is derived). Other proteins capable of specificallybinding immunoglobulin constant region, such as protein A or protein Gmay also be used as the labeled agent. These proteins exhibit a strongnon-immunogenic reactivity with immunoglobulin constant regions from avariety of species (see, e.g., Kronval et al., J. Immunol. 111:1401-1406 (1973); Akerstrom et al., J. Immunol. 135: 2589-2542 (1985)).The labeling agent can be modified with a detectable moiety, such asbiotin, to which another molecule can specifically bind, such asstreptavidin. A variety of detectable moieties are well known to thoseskilled in the art.

[0280] Throughout the assays, incubation and/or washing steps may berequired after each combination of reagents. Incubation steps can varyfrom about 5 seconds to several hours, preferably from about 5 minutesto about 24 hours. However, the incubation time will depend upon theassay format, antigen, volume of solution, concentrations, and the like.Usually, the assays will be carried out at ambient temperature, althoughthey can be conducted over a range of temperatures such as 10° C. to 40°C.

[0281] Non-Competitive Assay Formats

[0282] Immunoassays for detecting or quantitating a material (e.g., anautoinducer) in samples may be either competitive or noncompetitive.Noncompetitive immunoassays are assays in which the amount of antigen isdirectly measured. In one preferred “sandwich” assay, for example, ananti-autoinducer antibody can be bound directly to a solid substrate onwhich they are immobilized. These immobilized antibodies then capturethe autoinducer present in the test sample. The autoinducer is thusimmobilized and then bound by a labeling agent, such as a secondautoinducer antibody bearing a label. Alternatively, the second antibodymay lack a label, but it may, in turn, be bound by a labeled thirdantibody specific to antibodies of the species from which the secondantibody is derived. The second or third antibody is typically modifiedwith a detectable moiety, such as biotin, to which another moleculespecifically binds, e.g., streptavidin, to provide a detectable moiety.

[0283] Competitive Assay Formats

[0284] In competitive assays, the presence or amount of a material(e.g., an autoinducer) present in the sample is measured indirectly by,for example, measuring the amount of a known, added (exogenous)autoinducer displaced (competed away from an anti-autoinducer antibodyby the unknown autoinducer present in a sample. In one competitiveassay, a known amount of the autoinducer is added to a sample and thesample is then contacted with an antibody that specifically binds to theautoinducer. The amount of exogenous autoinducer bound to the antibodyis inversely proportional to the concentration of the autoinducerpresent in the sample. In a particularly preferred embodiment, theantibody is immobilized on a solid substrate. The amount of autoinducerbound to the antibody may be determined either by measuring the amountof autoinducer present in a autoinducer/antibody complex, oralternatively by measuring the amount of remaining uncomplexed protein.The amount of autoinducer can be detected by providing a labeledautoinducer molecule.

[0285] A hapten inhibition assay is another preferred competitive assay.In this assay a known autoinducer is immobilized on a solid substrate. Aknown amount of anti-autoinducer antibody is added to the sample, andthe sample is then contacted with the immobilized autoinducer. Theamount of anti-autoinducer antibody bound to the known immobilizedautoinducer is inversely proportional to the amount of autoinducerpresent in the sample. Again, the amount of immobilized antibody may bedetected by detecting either the immobilized fraction of antibody or thefraction of the antibody that remains in solution. Detection may bedirect where the antibody is labeled or indirect by the subsequentaddition of a labeled moiety that specifically binds to the antibody asdescribed above.

[0286] Cross-Reactivity Determinations

[0287] Immunoassays in the competitive binding format can also be usedfor cross-reactivity determinations for a material (e.g., anautoinducer). For example, a first autoinducer can be immobilized to asolid support. Other autoinducers, are added to the assay so as tocompete for binding of the antisera to the immobilized autoinducer. Theability of the added autoinducer(s) to compete for binding of theantisera to the immobilized autoinducer is compared to the ability ofthe first autoinducer to compete with itself. The percentcross-reactivity for the above autoinducers is calculated, usingstandard calculations. Those antisera with less than 10%cross-reactivity with each of the added autoinducers are selected andpooled. The cross-reacting antibodies are optionally removed from thepooled antisera by immunoabsorption with the added autoinducers, e.g.,related structural homologs.

[0288] The immunoabsorbed and pooled antisera are then used in acompetitive binding immunoassay as described above to compare a secondautoinducer, thought to be substantially structurally equivalent, to theimmunogen autoinducer. In order to make this comparison, the twoautoinducers are each assayed at a wide range of concentrations and theamount of each autoinducer required to inhibit 50% of the binding of theantisera to the immobilized autoinducer is determined. If the amount ofthe second protein required to inhibit 50% of binding is less than 10times the amount of the first autoinducer that is required to inhibit50% of binding, then the second protein is said to specifically bind tothe polyclonal antibodies generated to the respective autoinducerimmunogen.

[0289] Reduction of Non-Specific Binding

[0290] One of skill in the art will appreciate that it is oftendesirable to minimize non-specific binding in immunoassays.Particularly, where the assay involves an antigen or antibodyimmobilized on a solid substrate it is desirable to minimize the amountof non-specific binding to the substrate. Means of reducing suchnon-specific binding are well known to those of skill in the art.Typically, this technique involves coating the substrate with aproteinaceous composition. In particular, protein compositions such asbovine serum albumin (BSA), nonfat powdered milk, and gelatin are widelyused with powdered milk being most preferred.

[0291] Labels

[0292] The particular label or detectable group used in the assay is nota critical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and calorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

[0293] The label may be coupled directly or indirectly to the desiredcomponent of the assay according to methods well known in the art. Asindicated above, a wide variety of labels may be used, with the choiceof label depending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

[0294] Non-radioactive labels are often attached by indirect means.Generally, a ligand molecule (e.g., biotin) is covalently bourid to themolecule. The ligand then binds to another molecules (e.g.,streptavidin) molecule, which is either inherently detectable orcovalently bound to a signal system, such as a detectable enzyme, afluorescent compound, or a chemiluminescent compound. The ligands andtheir targets can be used in any suitable combination with antibodiesthat recognize the autoinducer, or secondary antibodies that recognizeanti-autoinducer antibodies.

[0295] The molecules can also be conjugated directly to signalgenerating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, or oxidotases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see, U.S. Pat.No. 4,391,904.

[0296] Means of detecting labels are well known to those of skill in theart. Thus, for example, where the label is a radioactive label, meansfor detection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the-fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

[0297] Some assay formats do not require the use of labeled components.For instance, agglutination assays can be used to detect the presence ofthe target antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

[0298] In a further preferred embodiment, the instant invention providesa method of monitoring the amount of autoinducer in a patient treatedwith an agent that inhibits the growth of an organism producing theautoinducer. The method includes the steps of: (a) providing a samplefrom the patient treated with the growth inhibiting agent; (b)contacting the sample with an antibody that specifically binds to anautoinducer; and (c) determining the amount of autoinducer in thepatient sample by detecting the antibody and comparing the amount ofantibody detected in the patient sample to a standard curve, therebymonitoring the amount of autoinducer in the patient.

[0299] In a preferred embodiment, the method is used to optimize thedosage of the growth inhibiting agent, thus, the method further includesthe step of adjusting the dose of the growth inhibiting agentadministered to the patient after examining the results of the assay.

[0300] Both of the above-described embodiments of the present inventionare useful in a number of methods of clinical and diagnostic relevance.Thus, in a preferred embodiment, the invention provides a method ofdetecting an autoinducer in a sample derived from a cultured cell, apatient sample and combinations thereof. Presently preferred patientsamples are blood samples, although other samples can be examined usingthe methods of the invention. Human samples are also preferred and amongsamples from humans those derived from patients with cystic fibrosis anda microbial infection are presently preferred.

[0301] Treatment of Diseases

[0302] In another aspect, the present invention provides a method fortreating or preventing a disease in a subject. The method includesadministering to the subject an amount of a composition of the inventionthat is sufficient to treat or prevent the disease. Compositions of theinvention appropriate for use in this aspect include, but are notlimited to, autoinducer analogues, antibodies, immunogenic conjugatesand combinations thereof. Moreover, in a preferred embodiment, thecompounds of the invention are conjugated with a bioactive agent.

[0303] The compositions of the present invention are of particular usein the treatment or prevention of diseases that are associated withorganisms that make use of autoinducers as a component of aquorum-sensing signal mechanism. Quorum sensing is a mechanism that isused by species, such as Gram-negative bacteria to inducedifferentiation and biofilm development (Fuqua et al, J. Bacteriol. 176:269 (1994); Fuqua et al., Ann. Rev. Microbiol. 50: 727 (1996)).“Biofilm,” as used herein, refers to a protected mode of growth of amicroorganism. The biofilm is a structured environment containingchannels through which nutrients can flow. A biofilm can be formed bymembers of the same species or one species can coaggregate with multiplepartners. The other partners can also aggregate with other partners,leading to the formation of a dense bacterial plaque (Whittaker et al,Annu. Rev. Microbiol. 50: 513 (1996); DeBeer et al., Biotech. Bioeng.44: 636 (1994)).

[0304] The organization of microorganisms into a biofilm has been foundto be modulated by signaling molecules, such as acylhomoserine lactoneproduced by individual cells. At a critical cell density, the signalmolecules accumulate to such a degree that they trigger the expressionof specific sets of genes, which have been implicated in the formationof biofilms.

[0305] Biofilms are of interest in medicine, because the organization ofa microbial colony in a host into a biofilm profoundly affects theaccess to much of the colony of exogenous therapeutic agents and thehost immune system (Cheema et al, J. Pharm. Pharmacol. (suppl. 38): 53P(1986); Gordon et al., J. Antimicrob. Chemother. 22: 667 (1988)). Thus,a method of interfering with the quorum-sensing mechanism and preventingor lessening the extent of biofilm formation, allows the microbialinfection to be more effectively treated using exogenous agents and alsoallows components of the host immune system improved access to themembers of the microbial colony.

[0306] In another preferred embodiment, the invention provides a methodfor preventing or disrupting the formation of a biofilm, the methodcomprising contacting a microbial culture capable of forming a biofilmwith a composition of the invention.

[0307] In a preferred embodiment of each of the above-describedembodiments of the invention, the instant method is used as a componentof the treatment of a microbial infection accompanying cystic fibrosis,such as P. aeruginosa.

[0308] Thus, in another preferred embodiment, the method is used todisrupt or prevent formation of a biofilm associated with an implantedmedical device, such as a catheter or stent (Stickler et al., Appl.Environ. Microbiol. 64: 3486 (1998)).

[0309] Regulation of Gene Expression

[0310] In another preferred embodiment, the present invention provides amethod of controlling autoinducer responsive gene expression in amicroorganism. The method includes contacting the microorganism with acompound or composition of the invention is an amount effective tocontrol the gene expression. The control exercise can be to decrease,but is preferably used to increase, gene expression. The organismsconcerned include bacteria, both Gram-positive and Gram-negative, yeastand fungi. This control technique preferably involves the use ofmicroorganisms that are not themselves capable of producing anautoinducer, but which are capable, in the presence of an exogenousautoinducer, of expressing a gene, preferably in an easily detectablemanner. The dosage of a compound of the invention necessary to affectgene expression can be ascertained by monitoring the gene expressionusing art recognized methods. Methods of regulating gene expression bythe application of endogenous autoinducers and detecting the regulationof the gene expression are described in Bycroft et al., U.S. Pat. No.5,593,827 and Salmond et al., U.S. Pat. No. 5,821,077, for example.

[0311] In another preferred embodiment, gene expression is decreased bycontacting the microorganism with an antibody of the invention in anamount effective to control said gene expression.

[0312] The materials and methods of the present invention are furtherillustrated by the examples, which follow. These examples are offered toillustrate, but not to limit the claimed invention.

EXAMPLES

[0313] The following Examples describe the synthesis andcharacterization of two of the reactive autoinducer analogues of theinvention. Example 1 sets for the synthesis of6-{N-((3S)-2-oxo(3,3,4,5-trihydrofuryl))-carbamoyl}-hexanoic acid (4).Example 2 sets forth the synthesis of13-{N-((3S)-2-oxo(3,3,4,5-trihydrofuryl))-carbamoyl}-11-oxoxtridecanoicacid (13).

Example 1

[0314] The following example sets forth the synthesis andcharacterization of Compound 4.

[0315] 1.1 Cyclization of Adipic Acid to Produce Compound 1

[0316] Adipic acid (100 g, 0.68 moles) and acetic anhydride (400 mL)were combined and refluxed for 14 hours. The mixture was concentrated toan amber oil on a rotary evaporator. The product was isolated by vacuumdistillation of the amber oil using an approximately 3 inch Vigreauxcolumn. Two fractions were collected. Fraction 1 was collected between110-125° C. and contained 36.8 g of material. Fraction 2 was collectedbetween 125-140° C. and contained 12.2 g. The extracts were combined toproduce a nominally pure material (49 g, 56%).

[0317] 1.2 Acylation of Benzyl Alcohol with Compound 1

[0318] Anhydride 1 (49.05 g, 0.38 moles) was added to a solution ofbenzyl alcohol (41.4 g, 0.38 moles) in dichloromethane (380 mL). Notemperature change was noted during the addition. The mixture wasstirred at room temperature, refluxed for 2 hours and then returned toroom temperature, where it was maintained overnight. Compound 2 wasisolated by distillation on a Kugelrohr apparatus. The fraction comingover at 160-175° C. at 0.1 mm was collected (45.1 g, 50%).

[0319] 1.3 Acylation of Homoserine Lactone with Compound 2

[0320] Under a N₂ atmosphere, L-homoserine lactone hydrobromide (10 g,55 mmol), mono-benzyladipate (2), pyridine (50 mL) and EDC hydrochloride(11.5 g, 60 mmol) were combined in dichloromethane (100 mL) in a 500 mLround-bottomed flask with cooling (˜0° C.). The resulting mixture wasstirred for 18 hours. The mixture was cooled to ˜0° C. and ice cold 15%citric acid solution was added. The citric acid layer was separated andextracted with dichloromethane (1×100 mL). The extract was combined withthe remaining dichloromethane reaction solution and the combineddichloromethane solutions were washed with brine, dried over sodiumsulfate, filtered and concentrated in vacuum, yielding 17.4 g (98.8%) ofthe crude product, which was slurried in isopropyl acetate to produce anoff-white solid. The solid was collected and dried under high vacuum at30° C., to produce 8.5 g (48.3%) of the desired product.

[0321] 1.4 Debenzylation of Compound 3

[0322] Compound 3 (5 g, 15.6 mmol) was suspended in ethyl acetate undera N₂ atmosphere. 10% Pd/C was added (5 g) and the N₂ atmosphere wasexchanged for a H₂ atmosphere, maintained using a balloon of H₂. Theresulting suspension was stirred vigorously. Because of productdepositing onto the catalyst, the suspension became unstirrable afterapproximately 1 hour. N₂ was bubbled through the reaction mixture toremove the H₂. Using an addition funnel, methanol (250 mL) was addedunder N₂ and the resulting mixture was stirred under N₂ for 10 minutes.The mixture was filtered through cellulose to remove the catalyst. Thecolorless filtrate was concentrated to afford an off-white so lid (3.8g). The solid was dissolved into hot acetontrile (50 mL), filteredthrough cellulose and reheated to dissolve the product. The clearsolution was allowed to cool slowly. The resulting needles werecollected and dried at 0.1 mm Hg affording the desired product. MP:140-141° C. Optical rotation: [α]_(D()25° C.)=−27.0° (c=1, methanol).Mass spectrum (m/z): 228 (M⁻¹). Elemental analysis: calculated: C 52.40;H 6.60; N 6.11. Found: C 52.51; H 6.53; N 6.21. ¹H NMR (DMSO) 8.35 (d,1H); 4.5 (m, 1H); 4.2 (m, 1H); 4.3 (m, 1H); 2.1-2.2 (m, 4H); 1.5 (m,4H). TLC (47.5:47.5:5.0 EtOAc/hexanes/AcOH), R_(f)=0.09.

Example 2

[0323] The following example sets forth the synthesis andcharacterization of Compound 13.

[0324] 2.1 Benzylation of Undecelinic Acid to Form Compound 4

[0325] Undecelinic acid (105.5 g, 1.05 moles) was placed in a 3-neckround bottomed flask with dimethylformamide (500 mL) and potassiumcarbonate (249.0 g, 1.8 moles) and was stirred overnight while in a N₂atmosphere. Benzyl bromide (100 g, 0.545 moles) was then added over 1hour after which the solution was stirred for 2 hours. TLC (1:12;isopropyl ether/hexane on SiO₂/glass) showed that the undecelinic acidhad been consumed.

[0326] The product solution was then filtered via Celite, washed twicewith water (2×300 mL) and the organic phases extracted with hexane(2×700 mL). The combined product solution was then dried with magnesiumsulfate, filtered and weighed to give 177 g (61% crude yield). ¹H NMRshowed that the product solution was benzylated undecelinic acid.

[0327] 2.2 Hydroboration of Compound 5 to Form Compound 6

[0328] Compound 5 (20.0 g, 72.9 mmol) and tetrahydrofuran (50 mL) werecombined in a 3-neck 250 mL round-bottomed flask under an N₂ atmosphere.The solution was cooled to 3° C. and 1M BH₃/THF complex (36.4 mL) wasadded dropwise over twenty minutes as the solution warmed from 3° C. to15° C. The solution was again cooled to 3° C. and upon removal from theice bath, stirred magnetically for three hours. TLC (1:9 ethylacetate/hexanes; developed with potassium permanganate) showed that thestarting material had been consumed.

[0329] 10% Sodium hydroxide (14 mL, 1-4° C.) was added over 4 minutes,35% hydrogen peroxide (11 mL, 3-23° C.) was added over 25 minutes andthe solution was allowed to warm to 17° C. over 30 minutes. Hydrochloricacid (100 mL, 1N) was added to the solution and the solution wasstirred.

[0330] The solution was poured into a separatory funnel and water (50mL) was added. The product was extracted with hexanes (2×60 mL) and thenwith ethyl acetate (1×75 mL). The combined organic layers were driedwith magnesium sulfate. The product was filtered and the solvents wereremoved under vacuum at 37° C., resulting in a clear oil. The oil wasdissolved in hexanes (100 mL) and ethyl acetate (4 mL). Seed crystalswere added and the solution was stirred in an ice bath for 1 hour. Theproduct was filtered and washed with cold hexanes (20 mL). Theextraction process was repeated to produce a second crop and a total of14.46 g of product (68% yield) was recovered. ¹H NMR of the crudematerial showed that the material was predominantly compound 6.

[0331] 2.3 Oxidation of Compound 6 to Produce Compound 7

[0332] Compound 6 (14.27 g, 48.8 mmol) was dissolved indimethylformamide (120 mL) in a 250 mL round-bottom flask. Pyridiniumdichromate (60.59 g, 161.0 mmol) was added portion-wise into thesolution and the solution was stirred magnetically overnight. TLC (1:19sodium hydroxide/dichloromethane; developed with phenylmercuric acetate)after 17 hours showed that the starting materials had been consumed.

[0333] The mixture was poured into water (600 mL) and extracted withtert-butyl methyl ether (2×600 mL). The product solution was dried withmagnesium sulfate and the solvents were removed under vacuum at 35° C.The product mixture was put on a high vacuum for 4 days to give 14.02 g(94% yield) of a lavender semi-solid. ¹H NMR analysis confirmed thatcompound 7 had been produced.

[0334] 2.4 Chlorination of Compound 7 to Give Compound 8

[0335] Compound 7 (11.05 g, 36.1 mmol) was dissolved in toluene (120 mL)in a 500 mL round-bottomed flask. The solution was cooled toapproximately 0° C. and oxalyl chloride (7.87 mL, 90.2 mmol) was addedover 5 minutes. The solution was stirred for overnight and afterapproximately 16 hours a pink oil settled in the bottom of the flask.

[0336] The pink oil was pipetted out into a small flask and discarded.After the solvents were removed from the remaining fluid under a vacuumat 40° C., a yellow oil with a small amount of solid was recovered andweighed to 12.59 g. ¹H NMR analysis confirmed that compound 7 hadformed.

[0337] 2.5 Preparation of Compound 9

[0338] A 500 mL 3-neck flask was equipped with an addition funnel, aircondenser, mechanical stirrer and N₂ line. The flask was charged with60% sodium hydride (2.48 g, 127.6 mmol) in mineral oil, which waswashed/decanted with hexanes (2×20 mL). Tetrahydrofuran (80 mL) wasadded to the flask. Di-tert-butyl propadioic acid (20.0 g, 92.5 mmol),which had been dissolved in tetrahydrofuran (40 mL), was added to thesuspension dropwise over 1 hour. The solution was stirred for another1.5 hours. tert-Butyl bromoacetate (11.15 g, 74.0 mmol) was suspended intetrahydrofuran (50 mL) and then added to the flask dropwise over 50minutes. A white precipitate formed. TLC analysis (2:8 hexanes/ether;developed with potassium permanganate) showed that starting materialshad been consumed.

[0339] The product solution was poured into saturated, aqueous ammoniumchloride (100 mL). Water (50 mL) and hexanes (150 mL) were added to theproduct solution and the flask was shaken to separate layers. Theorganic layer was removed. The aqueous layer was again extracted usinghexanes (150 mL). The organic layers were combined, dried with sodiumsulfate overnight and filtered. The solvents were removed under a vacuumat 39° C. and the product weighed to give 30.93 g of crude product.

[0340] The crude product was purified using flash SiO₂ chromatographyunder the following conditions: Solvent Amount petroleum ether* 50 mL 2:98 ether/petroleum ether 6 L  3:97 ether/petroleum ether 2 L  5:95ether/petroleum ether 2 L  7:93 ether/petroleum ether 2 L 15:85ether/petroleum ether 1 L

[0341] TLC in 1:20 ether/petroleum ether and developed with potassiumpermanganate confirmed all starting materials had been consumed.Further, ¹H NMR analysis showed that compound 9 had formed.

[0342] 2.6 Synthesis of Compound 10

[0343] A 500 mL flask was flushed with N₂ and charged with 60% sodiumhydride in mineral oil (1.59 g). The sodium hydride was washed/decantedwith hexanes (2×20 mL). Tetrahydrofuran (60 mL) was added to the flask.Compound 9 (11.92 g, 36.1 mmol) was suspended in tetrahydrofuran (65 mL)and then added to the flask dropwise over 0.5 hour. The solution wasthen stirred for 3 hours to afford a thin suspension. Compound 8 (11.7g, 36.1 mmol), which had been suspended in tetrahydrofuran (65 mL), wasthen added to the flask over 6 minutes. After 3.5 hours, TLC (2:8 ethylacetate/hexanes; developed with potassium permanganate) of the productsolution showed only a trace of starting material compound 8.

[0344] The product solution was then poured into saturated, aqueousammonium chloride (80 mL) and water (20 mL) was added. Hexanes (225 mL)was added, the flask was agitated to produce layers and the hexaneslayer was removed. The aqueous layer was extracted with hexanes (2×200mL). The organic layers were pooled, dried with magnesium sulfate andthe solvents removed under vacuum at 42° C. to give 22.2 g of a yellowoil (99% yield).

[0345] 2.7 Synthesis of Compound 11.

[0346] Compound 10 (22.0 g, 35.5 mmol) was dissolved in toluene (210 mL)in a 500 mL round-bottomed flask. p-Toluenesulfonic acid (0.43 g, 2.41mmol) was added to the flask and the resulting solution was heated undera N₂ atmosphere and allowed to reflux overnight. TLC (2:8 ethylacetate/hexanes; developed with potassium permanganate) of the solutionshowed that the starting material had been consumed.

[0347] The solution was washed with an aqueous buffer (125 mL) at pH 3and with saturated, aqueous sodium chloride (125 mL, pH=3). The aqueousphase was extracted with tert-butyl methyl ether (2×400 mL). Thecombined organic phase was washed with water (6×100 mL) until the pH ofthe water washes approached neutrality. The product solution was driedwith sodium sulfate and the solvents removed under vacuum at 47° C.,leaving a slurry. The product was then refluxed with hexanes (50 mL) andtert-butyl methyl ether (50 mL), air dried and cooled in an ice bath torecrystallize the product. The product was rinsed with hexanes (30 mL),air dried, and weighed to give 6.0 g of product material. ¹H NMR of theproduct indicated that compound 10 had formed.

[0348] 2.8 Synthesis of Compound 12

[0349] A 250 mL three-necked round-bottomed flask was charged withcompound 11 (5.47 g, 15.1 mmol) to which dichloromethane (50 mL) and1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.04 g,15.8 mmol) were added under a N₂ atmosphere. The resulting dark ambersolution was stirred at room temperature for 2.5 hours. Homoserinelactone hydrobromide (2.75 g, 15.1 mmol) was mixed with Hunig's Base(2.63 mL) and dichloromethane (55 mL) and added to the reaction solutionvia an addition funnel and stirred overnight. TLC (1:19 sodiumacid/dichloromethane, 2 drops of acetic acid) of the resulting productafter 18 hours showed the starting materials had been consumed.

[0350] The solution was poured into a mixture of ice water (150 ml) and2N hydrochloric acid (11 mL). Saturated, aqueous sodium chloride (50mL), hexanes (50 mL) and dichloromethane (50 mL) were added to separatethe layers and the aqueous phase was drawn off with dichloromethane(2×110 mL). The organic phase was washed with a saturated, aqueoussodium chloride/water mixture (1:1, 3×20 mL). The product solution wasdried with sodium sulfate, filtered slowly through a silica pad (232 g),eluted with 90% ethyl acetate/hexanes (500 mL) and the solvents wereremoved under vacuum at 41° C. and the white, solid product was weighedto 4.28 g (64% yield).

[0351] 2.9 Debenzylation of Compound 12 to Form Compound 13

[0352] A 2L 3-necked flask was equipped with a mechanical stirrer andflushed with N₂. A palladium/carbon catalyst (10%, 0.99 g) was added tothe flask. Compound 12 was dissolved in ethanol (750 mL) anddichloromethane (30 mL), and then stirred until the solid was completelydissolved. The solution was added to the flask and hydrogen gas wasbubbled through the solution via a glass frit. After 2 hours, a TLC (1:9sodium hydroxide/dichloromethane; developed with potassium permanganate)was taken and it was determined that no starting materials remained. Thesolution was filtered and the solvents removed under vacuum at 40° C.Thereafter, the product was placed under a vacuum for 2 hours to provide3.11 g (95% yield) of a white, solid product.

[0353] The product was recrystallized by dissolving 2.98 g of theproduct in isopropyl acetate (350 mL). The solution was suction filteredand then gently stirred at room temperature overnight. The product wasfiltered to remove a small amount of off-white solid. The filtrate wascooled in an ice bath for 1 hour to complete the precipitation and theproduct isolated by filtration. The product was dried in a vacuum oven(room temperature) for 4 hours affording 1.60 g of product (54% yield).TLC (1:9 sodium hydroxide/dichloromethane; developed with potassiumpermanganate) gave R_(f)=0.40.

[0354]¹H NMR showed that the recovered product was indeed compound 13.

Example 3

[0355] Example 3 sets forth the preparation of conjugates to KLA andBSA, the immunization schedule in rabbits and the results of antibodytiter measurements. The N-acyl derivative formed in Example I wasconjugated by the method of Anderson et al. J. Am Chem Soc. 86:1839-1844 (1964)) with slight modification. A solution of Compound 4(10mg, 43.6 μmol) NHS (5.0 mg, 43.6 μmol) and DCC (9.0 mg, 43.6 μmol) in0.125 mL dry dimethylformamide was stirred for four hours at roomtemperature. The precipitated dicyclohexylurea was filtered out anddiscarded. 80 μL of the filtrate was added into 20 mg KLH and theremaining 20 μL was added into 10 mg of either BSA or KLH in 1.0 mL eachof 0.1M bicarbonate buffer pH 8.25. After coupling with gentle agitationon a shaker for two hours, unconjugated 4 was separated from theconjugates by passing through Sephadex G25 equilibrated with PBS pH 7.2.The protein peak was collected and set aside for immunization and ELISAscreening for the antisera.

[0356] The conjugate of 4 was injected into 3 rabbits each at monthlyintervals to generate polyclonal antibodies. After 3 injections (3months) the rabbits were bled and the antibody titer measured by ELISA.

[0357] Figure X below shows the serial dilutions of the antisera of eachrabbit. The dilutions that reduces the ELISA by 50% are,respectively, >1:25,600 for rabbit R263, between 1:12,800 and. 1:25,600for R261 and between 1:12,800 and 1:25,600 for R262. Similar resultswere obtained with 13.

[0358] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to included within the spirit and purview of thisapplication and are considered within the scope of the appended claims.All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes.

What is claimed is:
 1. A compound having the structure:

wherein, R¹ is a member selected from —H, —OH, and (═O); R² is a memberselected from H, reactive functional groups, alkyl groups terminallysubstituted with a reactive functional group and internally substitutedalkyl groups terminally substituted with a reactive functional group; Xis a member selected from —O—, —S— and —NH—; and X¹ and X² are membersindependently selected from O and S.
 2. The compound according to claim1, wherein R² is an internally substituted alkyl group terminallysubstituted with a reactive functional group.
 3. The compound accordingto claim 2, wherein the alkyl group is internally substituted with afunctional group that is a member selected from —OH, (═O) andcombinations thereof.
 4. The compound according to claim 1, wherein thereactive functional group is a member selected from —OR³, —NHR⁴, —COR⁵,—SH and —CH₂X³ wherein, —OR³ is a member selected from hydroxy, alkylsulfonate and aryl sulfonate groups; R⁴ is a member selected from H,C₁-C₆ alkyl, C₁-C₆ substituted alkyl, aryl and substituted aryl groups;R⁵ is a member selected from H, X³ and —OR⁶, wherein R⁶ a memberselected from alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclyl and substitutedheterocyclyl groups; and X³ is a halogen.
 5. The compound according toclaim 1, wherein the compound is a single stereoisomer.
 6. The compoundaccording to claim 4, wherein R³ is

wherein, R⁸ is a member selected from alkyl, substituted alkyl, aryl andsubstituted aryl groups.
 7. The compound according to claim 1 whereinthe alkyl and the internally substituted alkyl groups are membersselected from C₁-C₂₀ saturated straight-chain, C₁-C₂₀ saturatedbranched-chain, C₁-C₂₀ unsaturated straight-chain, C₁-C₂₀unsaturated-branched-chain alkyl and internally substituted alkylgroups.
 8. The compound according to claim 7, wherein the alkyl andinternally substituted alkyl groups are members selected from C₅-C₁₀saturated straight-chain, C₅-C₁₀ saturated branched-chain, C₅-C₁₀unsaturated straight-chain, C₅-C₁₀ unsaturated branched-chain alkyl andinternally substituted alkyl groups.
 9. A compound according to claim 1,wherein R² has the structure: —(CH₂)_(n)—R⁷  (III) wherein, R⁷ areactive functional group; and n is a number from 1 to 20, inclusive.10. The compound according to claim 9, wherein n is a number from 2 to9, inclusive.
 11. A compound according to claim 1, wherein R² has thestructure:

wherein, R⁷ is a reactive functional group; and q and s are numbersindependently selected from 1 to 20, inclusive.
 12. The compoundaccording to claim 11, wherein s is a number from 2 to 9, inclusive. 13.A pharmaceutical formulation comprising a pharmaceutically acceptablecarrier and a compound according to claim 1, said reactive functionalgroup of said compound being covalently bound to a biologically activeagent.
 14. The pharmaceutical formulation according to claim 13, whereinsaid biologically active agent is a member selected from antibiotics,immune stimulators and combinations thereof.
 15. A compound having thestructure:

wherein, R¹ is a member selected from H, OH, and (═O); and. R² is amember selected from H, reactive functional groups, alkyl groupsterminally substituted with a reactive functional group and internallysubstituted alkyl groups terminally substituted with a reactivefunctional group, with the proviso that when R² is —OH, R¹ is a memberselected from OH, and (═O).
 16. The compound according to claim 15,wherein the reactive functional group is a member selected from —OR³,—NHR⁴, —COR⁵, SH and CH₂X³ wherein, —OR³ is a member selected fromhydroxy, and a species such that —OR³ is a leaving group; R⁴ is a memberselected from H, C₁-C₆ alkyl, C₁-C₆ substituted alkyl, aryl andsubstituted aryl groups; R⁵ is a member selected from H, halogen and—OR⁶, wherein R⁶ is species such that —OR⁶ is a leaving group; and X³ isa halogen.
 17. The compound according to claim 16, wherein R³ is

wherein, R⁸ is a member selected from alkyl, substituted alkyl, aryl andsubstituted aryl groups.
 18. The compound according to claim 16, whereinR⁶ is a member selected from alkyl, substituted alkyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclyl and substitutedheterocyclyl groups.
 19. The compound according to claim 15, wherein thealkyl and the internally substituted alkyl groups are members selectedfrom C₁-C₂₀ saturated straight-chain, C₁-C₂₀ saturated branched-chain,C₁-C₂₀ unsaturated straight-chain, C₁-C₂₀ unsaturated branched-chainalkyl and internally substituted alkyl groups.
 20. The compoundaccording to claim 19, wherein the alkyl and internally substitutedalkyl groups are members selected from C₅-C₁₀ saturated straight-chain,C₅-C₁₀ saturated branched-chain, C₅-C₁₀ unsaturated straight-chain,C₅-C₁₀ unsaturated branched-chain alkyl and internally substituted alkylgroups.
 21. A compound according to claim 15, wherein R² has thestructure: —(CH₂)_(n)—R⁷  (III) wherein, R⁷ is a reactive functionalgroup; and n is a number from 1 to 20, inclusive.
 22. The compoundaccording to claim 21, wherein n is a number from 2 to 9, inclusive. 23.The compound according to claim 15, wherein R² is a member selected fromthe group consisting of —COOH, —OH, —NH₂, and —SH.
 24. The compoundaccording to claim 21, wherein R⁷ is a member selected from the groupconsisting of —COOH, —OH, —NH₂, and —SH.
 25. A compound having astructure that is a member selected from:

wherein, m is a number selected from 1 to 20, inclusive; n is a numberfrom 0 to 20, inclusive; and Z is a reactive functional group.
 26. Thecompound according to claim 25, wherein m and n are numbersindependently selected from 2 to 9, inclusive.
 27. The compoundaccording to claim 25, wherein Z is a member selected from —NH₂, —COOH,—SH, and —OH.
 28. An immobilized compound comprising a solid support towhich is attached a molecule comprising the structure:

wherein, R¹ is a member selected from —H, —OH, and (═O); R⁹ is a memberselected from alkyl groups and substituted alkyl groups; X is a memberselected from —O—, —S— and —NH—; X¹ and X² are members independentlyselected from O and S.
 29. The immobilized compound according to claim28, wherein the solid support is a member selected from beads,particles, membranes, substantially planar surfaces and combinationsthereof.
 30. The immobilized compound according to claim 28, wherein thesolid support comprises a member selected from silica, metal, plasticand combinations thereof.
 31. The immobilized compound according toclaim 28, wherein R⁹ comprises a spacer moiety situated between themolecule and the solid support.
 32. The immobilized compound accordingto claim 31, wherein the spacer moiety is selected from C₆-C₃₀ alkylgroups, C₆-C₃₀ substituted alkyl groups, polyols, polyethers,polyamines, polyamino acids, polysaccharides and combinations thereof.33. The immobilized compound according to claim 31, wherein the spacermoiety comprises a cleavable moiety.
 34. The immobilized compoundaccording to claim 33, wherein the cleavable moiety is cleaved by amember selected from light, heat, oxidation, reduction, enzymaticaction, hydrolysis and combinations thereof.
 35. The immobilizedcompound according to claim 34, wherein the cleavable moiety is a memberselected from disulfides and esters.
 36. A method for isolating amicrobial receptor binding to a molecule comprising the formula:

wherein, R¹ is a member selected from —H, —OH, and (═O); R⁹ is a memberselected from alkyl groups and substituted alkyl groups; X is a memberselected from —O—, —S— and —NH—; X¹ and X² are members independentlyselected from O and S; the method comprising: contacting a microbialpreparation comprising the receptor with the immobilized compoundaccording to claim 28, thereby forming a complex between the receptorand the immobilized compound.
 37. The method according to claim 36,further comprising separating the complex from components of themicrobial preparation not comprising the receptor.
 38. The methodaccording to claim 37, further comprising disrupting the complex betweenthe immobilized compound and the receptor, thereby separating thereceptor from the immobilized compound.
 39. An immunogenic conjugatecomprising a target component comprising the structure:

wherein, R¹ is a member selected from —H, —OH, and (═O); R⁹ is a memberselected from alkyl groups and substituted alkyl groups; X is a memberselected from —O—, —S— and —NH—; and X¹ and X² are members independentlyselected from O and S.
 40. The immunogenic conjugate according to claim39, wherein the target component comprises the structure:

wherein, R¹ is a member selected from H, OH, and (═O); and R⁹ is amember selected from alkyl and substituted alkyl groups.
 41. Theimmunogenic conjugate according to claim 40, wherein the targetcomponent has the structure:

wherein, m is a number from 0 to 30, inclusive.
 42. The immunogenicconjugate according to claim 39 having the structure:

wherein, R¹ is a member selected from —H, —OH, and (═O), R⁹ is a memberselected from alkyl groups and substituted alkyl groups; X is a memberselected from —O—, —S— and —NH—; X¹ and X² are members independentlyselected from O and S; and P is a protein carrier.
 43. The immunogenicconjugate according to claim 42, wherein the protein carrier has amolecular weight of greater than or equal to 5000 daltons.
 44. Theimmunogenic conjugate according to claim 43, wherein the protein carrieris a member selected from albumin and hemocyanin.
 45. The immunogenicconjugate according to claim 39, wherein R⁹ comprises a spacer moietysituated between the target component and the protein carrier.
 46. Theimmunogenic conjugate according to claim 45, wherein the spacer moietyis selected from C₆-C₃₀ alkyl groups, C₆-C₃₀ substituted alkyl groups,polyols, polyethers, polyamines, polyamino acids, polysaccharides andcombinations thereof.
 47. The immunogenic conjugate according to claim45, wherein the spacer moiety comprises a cleavable moiety.
 48. Theimmunogenic conjugate according to claim 47, wherein the cleavablemoiety is cleaved by a member selected from light, heat, oxidation,reduction, enzymatic action, hydrolysis and combinations thereof. 49.The immunogenic conjugate according to claim 48, wherein the cleavablemoiety is a member selected from disulfides and esters.
 50. Apharmaceutical formulation comprising the immunogenic conjugateaccording to claim 39 and a pharmaceutically acceptable carrier.
 51. Thepharmaceutical formulation according to claim 50, wherein thepharmaceutical formulation is a vaccine effective for preventing orreducingmicrobial infection in a subject to whom the vaccine isadministered.
 52. An antibody that binds specifically to the immunogenicconjugate according to claim
 39. 53. An isolated nucleic acid encodingthe antibody according to claim
 52. 54. The isolated nucleic acidaccording to claim 53, further comprising a promoter operably linked tothe nucleic acid sequence encoding the antibody.
 55. An expressionvector comprising the nucleic acid according to claim
 53. 56. A hostcell comprising the expression vector according to claim
 55. 57. Theantibody according to claim 52, further comprising a member selectedfrom detectable labels, biologically active agents and combinationsthereof covalently attached to the antibody.
 58. The antibody accordingto claim 57, wherein the detectable label is a member selected from thegroup consisting of radioactive isotopes, fluorescent agents,fluorescent agent precursors, chromophores, enzymes and combinationsthereof.
 59. The antibody according to claim 58, wherein thebiologically active agent is a member selected from antibiotics, immunestimulators and combinations thereof.
 60. A pharmaceutical formulationcomprising the antibody according to claim 52 and a pharmaceuticallyacceptable carrier.
 61. A method for treating or preventing a disease ina subject caused by a microorganism, the method comprising administeringto the subject an amount of the antibody according to claim 52 effectiveto reduce or prevent the disease state.
 62. A method for treating orpreventing a disease in a subject caused by a microorganism, the methodcomprising administering to the subject an amount of the vaccineaccording to claim 51 effective to reduce or prevent the disease state.63. A method for treating or preventing a disease in a subject caused bya microorganism, the method comprising administering to the subject anamount of the immunogenic conjugate according to claim 39 effective toreduce or prevent the disease state.
 64. The method according to claim61, wherein the disease is a microbial infection.
 65. The methodaccording to claim 62, wherein said microbial infection accompaniescystic fibrosis.
 66. The method according to claim 74, wherein saidmicrobial infection has a causative agent comprising P. aeruginosa. 67.A method for preventing or disrupting the formation of a biofilm, themethod comprising contacting a microbial culture capable of forming abiofilm with an antibody according to claim
 52. 68. The method accordingto claim 67, wherein said biofilm comprises P. aeruginosa.
 69. Themethod according to claim 67, wherein said biofilm is associated with animplanted medical device.
 70. The method according to claim 67, whereinsaid biofilm is associated with an organ in vivo.
 71. A method forcontrolling autoinducer responsive gene expression in a microorganism,the method comprising contacting the microorganism with an antibodyaccording to claim 52 effective to control said gene expression.
 72. Amethod for controlling autoinducer responsive gene expression in amicroorganism, the method comprising contacting the microorganism withan antibody according to claim 51 effective to control said geneexpression.
 73. A method for controlling autoinducer responsive geneexpression in a microorganism, the method comprising contacting themicroorganism with an antibody according to claim 39 effective tocontrol said gene expression.
 74. The method according to claim 71,wherein the microorganism is bacteria.
 75. The method according to claim74, wherein said bacteria is P. aeruginosa.
 76. A library of compoundscomprising a structure according to Formula I:

wherein, R¹ is a member selected from —H, —OH, and (═O); R⁹ is a memberselected from alkyl groups and substituted alkyl groups; X is a memberselected from —S— and —NH—; X¹ and X² are members independently selectedfrom O and S, the library comprising a first compound according toFormula I and a second compound according to Formula I, wherein thefirst compound differs from the second compound in the identity of amember selected from R¹, R⁹, X, X¹, X and combinations thereof.
 77. Thelibrary according to claim 76, comprising at least 10 compounds.
 78. Thelibrary according to claim 77, comprising at least 100 compounds. 79.The library according to claim 78 comprising at least 1000 compounds.80. The library according to claim 79 comprising at least 100,000compounds.
 81. A method of detecting an autoinducer in a sample, themethod comprising the steps of: (a) contacting the sample with anantibody that specifically binds to the autoinducer; and (b) determiningwhether the sample contains the autoinducer, thereby detecting saidautoinducer.
 82. The method of claim 81, wherein the antibody is amonoclonal antibody.
 83. The method of claim 81, wherein the antibody isa polyclonal antibody.
 84. The method of claim 81, wherein the step ofdetermining whether the sample contains an autoinducer comprisesdetecting the antibody in an assay selected from the group consisting ofan ELISA assay, a western blot, an immunohistochemical assay, animmunofluorescence assay, and a real time imaging assay.
 85. The methodof claim 81, wherein the step of determining whether the sample containsan autoinducer further comprises quantitating the amount of autoinducerin the sample.
 86. The method of claim 81, wherein the antibody is boundto a solid substrate.
 87. The method of claim 81, wherein the sample isselected from the group consisting of a cultured cell, and a patientsample.
 88. The method of claim 87, wherein the patient sample is ablood sample.
 89. The method of claim 87, wherein the patient sample isfrom a human patient.
 90. The method of claim 81, wherein the antibodyis covalently linked to a detectable moiety.
 91. The method of claim 90,wherein the antibody is covalently linked to a member selected from abiotin moiety, a radioactive moiety, an enzyme moiety and combinationsthereof.
 92. A method of monitoring the amount of autoinducer in apatient treated with an agent that inhibits the growth of an organismproducing the autoinducer, the method comprising: (a) providing a samplefrom the patient treated with the growth inhibiting agent; (b)contacting the sample with an antibody that specifically binds to anautoinducer; and (c) determining the amount of autoinducer in thepatient sample by detecting the antibody and comparing the amount ofantibody detected in the patient sample to a standard curve, therebymonitoring the amount of autoinducer in the patient.
 93. The method ofclaim 92, further comprising the step of adjusting the dose of thegrowth inhibiting agent administered to the patient.
 94. The method ofclaim 92, wherein the sample is a blood sample.
 95. The method accordingto claim 94, wherein said blood sample is derived from a patient havingcystic fibrosis and an infection comprising P. aeruginosa.
 96. Themethod of claim 92, wherein the antibody is a inonoclonal antibody. 97.The method according to claim 92, wherein said antibody is a polyclonalantibody.
 98. The method of claim 92, wherein the antibody is covalentlylinked to a detectable moiety.
 99. The method of claim 98, wherein theantibody is covalently linked to a member selected from a biotin moiety,a radioactive moiety, an enzyme moiety and combinations thereof. 100.The method of claim 92, wherein the antibody is bound to a solidsubstrate.
 101. A method of isolating an autoinducer, the methodcomprising the steps of: (a) providing a sample comprising theautoinducer; (b) contacting the sample with an antibody thatspecifically binds to the autoinducer, thereby forming anautoinducer-antibody complex; and (c) isolating the autoinducer-antibodycomplex by isolating the antibody.
 102. The method of claim 101, whereinthe antibody is a monoclonal antibody.
 103. The method of claim 101,wherein the antibody is covalently linked to member selected from abiotin moiety, a radioactive moiety, an enzyme moiety and combinationsthereof.
 104. The method of claim 101, wherein the antibody is bound toa solid substrate.
 105. A method of detecting an antibody thatspecifically binds to an autoinducer, the method comprising the stepsof: (a) providing a sample; (b) contacting the sample with a peptidethat specifically binds to the antibody; and (c) detecting the antibody.106. The method of claim 105, wherein the step of detecting the antibodycomprises an ELISA assay.
 107. The method of claim 105, wherein thepeptide is bound to a solid substrate.
 108. A kit for detecting anautoinducer in a sample, the kit comprising: (a) an antibody that bindsspecifically to the autoinducer; (b) directions for using the antibodyto detect the autoinducer.